WO2013079606A1 - Automated dual stain of mirna and protein targets - Google Patents

Abstract

Particular disclosed embodiments concern an automated method for dually detecting and staining miRNA and protein targets. The disclosed method does not employ protease cell conditioning; rather the method comprises non-enzymatic cell conditioning, contacting a sample with a nucleic acid specific binding moiety and a protein specific binding moiety, and detecting the mi RNA target and protein target. Also disclosed is an apparatus for performing the disclosed method. Using the disclosed apparatus allows the method to be performed in a fully automated manner.

Description

AUTOMATED DUAL STAIN OF miRNA AND PROTEIN TARGETS

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/565,857, filed December 1, 2011, which is incorporated herein by reference in its entirety.

FIELD

The present invention concerns an automated method for dual staining miRNA and protein. Disclosed embodiments provide an automated diagnostic for visualizing the expression of the protein and the miRNA within a single tissue sample with anatomical context intact. Disclosed embodiments avoid certain deleterious process steps associated with manual staining techniques, such as enzymatic preconditioning of a tissue sample.

BACKGROUND

MicroRNAs (miRNAs) are short ribonucleic acid molecules (i.e. average of

22 nucleotides) that regulate biological information at the post-transcriptional level. miRNAs can, for example, bind to complementary sequences on target messenger RNA (mRNA) transcripts to regulate translation. Eukaryotic miRNAs are known to inhibit protein translation of a target mRNA. Given their small size and lack of 100% homology copies of miRNA can be produced in very high numbers, relative to mRNA. miRNAs also cause histone modification and DNA methylation of promoter sites, which affects the expression of target genes.

Based on early studies, miRNAs play a role in developmental regulation and cell differentiation in mammals, as well as cardiogenesis and lymphocyte development. In addition, miRNA are involved in other biological processes, such as hypoxia, apoptosis, stem cell differentiation, proliferation, inflammation, and response to infection. miRNA can be used to concurrently target multiple effectors of pathways involved in cell differentiation, proliferation and survival, key characteristics of oncogenesis. Several miRNAs have been linked to cancer. As a result, in-situ analysis of miRNA can be useful for cancer diagnosis and therapeutics, as miRNAs appear to act as oncogenes or tumor repressors. For example, many tumor cells have distinct miRNA expression patterns when compared with normal tissues. Studies using mice genetically altered to produce excess c-Myc - a protein with mutated forms implicated in several cancers - established that miRNA effects cancer

development. Mice engineered to produce surplus miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days. Leukemia can also be caused by increased expression of miRNA. It has been demonstrated that the differential measurement of miRNAs can be used to distinguish various types of cancers. The expression levels of miRNAs can also be used as a prognostic. For example, low miR-324a levels serve as a prognostic indicator of a poor survival rate, and high miR-185 levels or low miR-133b levels correlate with metastasis and poor survival in colorectal cancer.

miRNAs also play a role in heart function. Conditionally inhibiting miRNA expression in the murine heart has established that miRNAs play an essential role during heart development. miRNA expression levels of specific miRNAs change in diseased human hearts, which indicates involvement in cardiomyopathies. miRNAs also appear to regulate the nervous system, and neural miRNAs are involved at various stages of synaptic development. For example, miRNAs are involved in dendritogenesis (involving miR-132, miR-134 and miR- 124), synapse formation and synapse maturation (where miR-134 and miR-138 are thought to be involved). Altered miRNA expression levels also have been implicated in schizophrenia. Clearly, miRNAs are now known to have substantial biological effects.

Methods for detecting miRNA, as well as protein translated or otherwise regulated by miRNA, are highly desirable, particularly in automated methods for efficient and rapid detection. Prior methods for detecting miRNA do not detect both miRNA and its protein expression targets (potentially regulated by the miRNA) in the same sample. Previous methods typically require using protease- based cell conditioning to digest cellular components to expose nucleic acid targets. Furthermore, previous methods correlate levels of miRNA and protein levels using northern and western blots. Although these methods enable miRNA determination, they require distinct manual steps which are time consuming and prone to human error. Many of these manual steps do not translate well to automation. Further, molecular approaches that "grind and bind" the sample result in a loss of contextual information between the cellular structure and the various miRNA and proteins analyzed. While tissue-based approaches have been previously demonstrated, they lack the ability to multiplex (e.g. dual staining or greater) across numerous targets concurrently or lack complete automation. Additionally, these methods generally include an enzymatic step which often interferes with protein identification and/or cellular morphology.

As miRNAs play an important role in several biological processes, it is likely that they open a new avenue for therapeutic intervention and/or diagnostic analysis. Accordingly, a need exists in the field for a cost- and time-efficient automated method for detecting both a miRNA target and a protein, particularly for those combinations of miRNA and proteins that are biologically inter-related.

SUMMARY

Disclosed embodiments concern an automated method particularly suited for multiplexed detection of miRNA and proteins. In illustrative embodiments, the expression of the one or more proteins may be regulated by the miRNA. In another embodiment, the method enables the cellular context between the miRNA and the protein to be identified. The method may comprise, for example, using an automated system to apply to a tissue sample (a) reagents suitable for detecting a miRNA target, (b) reagents suitable for detecting a protein target, and (c) reagents suitable for staining the miRNA target and the protein target. One aspect of the present embodiments concerns using non-enzymatic cell conditioning, i.e. avoiding protease-based cell conditioning, to preserve the protein targets. A cell

conditioning step can involve treating the sample with a cell conditioning solution, such as a buffer having a slightly basic pH, including a Tris-based buffer having a pH from about 7.7 to about 9, at a temperature greater than ambient, such as from about 80 °C to about 95 °C. The automated method can detect the miRNA and protein targets simultaneously or sequentially, although better staining results typically are obtained by first detecting and staining the miRNA and then detecting and staining the protein target.

A more particular disclosed embodiment first comprises performing non- enzymatic cell conditioning on the sample. The sample is then contacted with a nucleic acid specific binding moiety selected for a particular miRNA target, followed by detecting the miRNA specific binding moiety. The sample is then contacted with a protein specific binding moiety selected for a protein target, followed by detecting the protein specific binding moiety. In certain embodiments, the nucleic acid specific binding moiety is a locked nucleic acid (LNA) probe conjugated to a detectable moiety, such as an enzyme, a fluorophore, a

luminophore, a hapten, a fluorescent nanoparticle, or combinations thereof. Certain suitable haptens are common in the art, such as digoxigenin, dinitrophenyl, biotin, fluorescein, rhodamine, bromodeoxyuridine, mouse immunoglobulin, or combinations thereof. Other suitable haptens were specifically developed by

Ventana Medical Systems, Inc., including haptens selected from oxazoles, pyrazoles, thiazoles, benzofurazans, triterpenes, ureas, thioureas, rotenoids, coumarins, cyclolignans, heterobiaryls, azoaryls, benzodiazepines, and

combinations thereof. Haptens can be detected using an anti-hapten antibody. In certain disclosed embodiments, the anti-hapten antibody is detected by an anti- species antibody-enzyme conjugate, wherein the enzyme is any suitable enzyme, such as alkaline phosphatase or horseradish peroxidase.

Particular embodiments involved contacting the sample with a digoxigenin- labeled, locked nucleic acid probe selected for the miRNA target. For these disclosed embodiments, the automated method further comprises contacting the sample with a mouse anti-digoxigenin antibody, contacting the sample with a conjugate comprising an anti-mouse antibody conjugated to alkaline phosphatase, and contacting the sample with an alkaline phosphatase substrate system.

For certain embodiments, the protein specific binding moiety may be a primary antibody. The primary antibody is detectable by a second specific binding moiety, such as a secondary anti-antibody conjugated to a detectable moiety, such as a hapten. For these embodiments, the method may further comprise contacting the sample with an anti-hapten antibody-enzyme conjugate, followed by contacting the sample with an enzyme substrate. In yet other embodiments, the automated method includes multi-color chromogenic detection of one or several miR A and one or several proteins.

Any suitable enzyme/enzyme substrate system can be used for the disclosed automated method. Working embodiments typically used alkaline phosphatase and horseradish peroxidase. If the enzyme is alkaline phosphatase, one suitable substrate is nitro blue tetrazolium chloride/(5-bromo-4-chloro-lH-indol-3-yl) dihydrogen phosphate (NBT/BCIP). If the enzyme is horseradish peroxidase, then one suitable substrate is diaminobenzidine (DAB). Other detection or signal amplification systems known in the art can be used with the automated system, such as tyramide-based detection. In other embodiments, one could use indirect fluorescence-based systems as well (e.g. organic dyes, quantum dots, or nanocrystals).

A particular embodiment of the automated method comprises

deparaffinizing the tissue sample, and then performing non-enzymatic cell conditioning. The sample is then contacted with a hapten-labeled LNA probe selected for the miRNA target. The sample is heated, and then cooled to a hybridization temperature below the T_m for the LNA probe. The sample is hybridized with the probe at the hybridization temperature for a period of time suitable for hybridization. The sample is then contacted with an anti-hapten antibody, an anti-antibody-enzyme conjugate, and then treated with an enzyme substrate suitable for visualizing the miRNA target. The sample is then contacted with a primary antibody selected for detecting the protein target. A secondary anti- antibody labeled with at least one hapten is then used to detect the primary antibody. The sample is then contacted with an anti-hapten antibody-enzyme conjugate, followed by an enzyme substrate suitable for visualizing the protein target. For certain working embodiments, the method further comprises applying a hematoxylin stain and a bluing reagent.

Kits suitable for practicing the method also are disclosed. A kit may comprise, for example, (a) a non-enzymatic cell conditioning solution, (b) reagents suitable for use in an automated system for detecting an miRNA target in the sample, such as a locked nucleic acid-hapten conjugate, an anti-hapten antibody- enzyme conjugate, and a substrate for the enzyme, and (c) reagents suitable for use in an automated system for detecting a protein target, such as a primary antibody, a secondary anti-antibody-enzyme conjugate, and an enzyme substrate. The kit may further comprise a hematoxylin stain and a bluing reagent

The disclosed methods are suitable for automated systems, such as Ventana Medical Systems, Inc.'s Discovery series of instruments. The process steps performed by such devices are controlled by software. Accordingly, disclosed embodiments also concern computer readable media comprising instructions for performing the disclosed embodiments of the automated method.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1(A)-(C) are photomicrographs showing serial sections of a breast cancer tissue stained using a dual staining procedure, as described herein, wherein (A) shows the dual detection of HER3 (detected with DAB) and miR-205 (detected with NBT/BCIP), (B) shows a control in which the dual staining procedure was done without miR-205 probe, and (C) shows a control in which the dual staining procedure was done without anti-HER3.

FIG. 2(A)-(B) are photomicrographs showing the results obtained using a dual stain protocol for miR-205 (detected with NBT/BCIP) and Bcl2 (detected with DAB) on (A) a first breast cancer tissue and (B) a second breast cancer tissue.

FIG. 3(A)-(D) are photomicrographs showing serial sections of a lung tissue stained using a dual staining procedure, as described herein, wherein (A) shows the dual detection of CRK (detected with DAB) and miR-126 (detected with NBT/BCIP), (B) shows a control in which the dual staining procedure was done without miR-126 probe, (C) shows a control in which the dual staining procedure was done without anti-CRK, and (D) shows a control in which the dual staining procedure was done without anti-CRK and using a scramble probe in place of the miR-126 probe. FIG. 4(A)-(B) are photomicrographs showing serial sections of a breast tissue stained using the dual staining procedure described herein without inclusion of the miR-205 probe, wherein (A) shows the deleterious effects of using a proteinase conditioning on a sample used to detect HER3 and (B) shows the substantial beneficial results obtained using a cell conditioning process comprising a buffer and heat preconditioning instead of proteinase pretreatment for detecting HER3.

FIG. 5(A)-(B) are photomicrographs showing serial sections of a breast tissue stained using the dual staining procedure described herein without inclusion of the anti-HER3 antibody, wherein (A) shows the deleterious effects of using a proteinase conditioning on a sample used to detect miR-205 probe and (B) shows the substantial beneficial results obtained using a cell conditioning process comprising of a buffer and heat preconditioning instead of proteinase pretreatment for detecting miR-205.

FIG. 6(A)-(B) are photomicrographs showing sections of a breast tissue stained using the dual staining procedure described herein without inclusion of a target-specific antibody reagent, wherein (A) is a negative control in which a scramble probe was used to demonstrate a lack of non-specific staining and (B) is a positive control in which a U6 probe was used to demonstrate that the sample was penetrated by the probe and confirming the ability to hybridize probes with miRNA available in the sample.

DETAILED DESCRIPTION

I. Introduction

In order to facilitate review of the various features and examples of this disclosure, certain specific term explanations are provided below. Several exemplary embodiments also are described as non-limiting examples that illustrate the scope and features of the invention. The section titles are not to be construed as limitations, but are provided to structure this specification.

II. Abbreviations and Definitions

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. It is further to be understood that all nucleotide sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides or other compounds are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Amp HQ: An enzyme-based target detection amplification kit utilizing a hydroxyquinoxiline (HQ) hapten in combination with an anti-HQ multimer, an HRP multimer, a chromogenic detection kit for enhancing the chromogenic signal in IH and ISH methodologies.

Amplification: Certain embodiments of the present invention allow a single target to be detected using plural visualization complexes, where the complexes can be the same or different, to facilitate identification and/or quantification of a particular target.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows.

Antibody: "Antibody" collectively refers to immunoglobulins or immunoglobulin-like molecules (including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10³ M^"1 greater, at least 10⁴ M^"1 greater or at least 10⁵ M^"1 greater than a binding constant for other molecules in a biological sample.

More particularly, "antibody" refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V_R) region and the variable light (V_L) region. Together, the V_R region and the V_L region are responsible for binding the antigen recognized by the antibody.

This includes intact immunoglobulins and the variants and portions of them well known in the art. Antibody fragments include proteolytic antibody fragments [such as F(ab')₂ fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as are known in the art], recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'₂ fragments, single chain Fv proteins ("scFv"), disulfide stabilized Fv proteins ("dsFv"), diabodies, and triabodies (as are known in the art), and camelid antibodies (see, for example, U.S. patent Nos. 6,015,695; 6,005,079-5,874,541; 5,840,526; 5,800,988; and 5,759,808). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, III); Kuby, J., Immunology, 3.sup.rd Ed., W.H. Freeman & Co., New York, 1997.

Antigen: A compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens. In one example, an antigen is a

Bacillus antigen, such as yPGA.

Automated: Refers to a method where one or more steps are executed by substantially mechanical, electro-mechanical, computer, and/or electronically controlled systems. It does not exclude some human intervention steps such as loading samples on slides and/or manually performing one or more of the features or steps described herein.

Avidin: Any type of protein that specifically binds biotin to the substantial exclusion of other small molecules that might be present in a biological sample. Examples of avidin include avidins that are naturally present in egg white, oilseed protein (e.g., soybean meal), and grain (e.g., corn/maize) and streptavidin, which is a protein of bacterial origin.

Binding affinity: The tendency of one molecule to bind (typically non- covalently) with another molecule, such as the tendency of a member of a specific binding pair for another member of a specific binding pair. A binding affinity can be measured as a binding constant, which binding affinity for a specific binding pair (such as an antibody/antigen pair or nucleic acid probe/nucleic acid sequence pair) can be at least 1 x 10⁵ M^"1, such as at least 1 x 10⁶ M^"1, at least 1 x 10⁷ M^"1 or at least 1 x 10⁸ M^_1.

Carrier: A molecule to which a hapten or an antigen can be bound.

Carrier molecules include immunogenic carriers and specific-binding carriers.

When bound to an immunogenic carrier, the bound molecule may become immunogenic. Immunogenic carriers may be chosen to increase the

immunogenicity of the bound molecule and/or to elicit antibodies against the carrier, which are diagnostically, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T-cell dependence (Pozsgay et al, PNAS 96:5194-97, 1999; Lee et al, J. Immunol. 116: 1711-18, 1976; Dintzis et al, PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers, which can be natural (for example, proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached. Specific binding carriers can by any type of specific binding moiety, including an antibody, a nucleic acid, an avidin, a protein-nucleic acid.

Examples of suitable immunogenic carriers are those that can increase the immunogenicity of a hapten and/or help elicit antibodies against the hapten which are diagnostically, analytically, and/or therapeutically beneficial. Useful carriers include polymeric carriers, which can be natural (such as proteins like ovalbumin or keyhole limpet hemocyanin) or derived from a natural polymer isolated from any organism (including viruses), semi-synthetic or synthetic materials containing one or more functional groups, for example primary and/or secondary amino groups, azido groups, hydroxyl groups, or carboxyl groups, to which a reactant moiety can be attached. The carrier can be water soluble or insoluble, and in some embodiments is a protein or polypeptide. Carriers that fulfill these criteria are generally known in the art (see, for example, Fattom et al, Infect. Immun. 58:2309- 12, 1990; Devi et al, PNAS 88:7175-79, 1991; Szu et al, Infect. Immun. 59:4555- 61, 1991; Szu et al, J. Exp. Med. 166: 1510-24, 1987; and Pavliakova et al, Infect. Immun. 68:2161-66, 2000).

Cell conditioning reagent: An aqueous solution useful for conditioning cell samples, such as prior to hybridization in methods of in situ hybridization. For example, cell conditioning reagents include those disclosed in U.S. patent application Ser. No. 09/800,689, filed Mar. 7, 2001, which is hereby incorporated by reference in its entirety.

Cell conditioning solution: A cell conditioning reagent. In one embodiment, cell conditioning solution comprises sodium citrate; citric acid; "cell conditioning preservative"; and nonionic detergent. In a preferred embodiment, the nonionic detergent is "cell conditioning detergent." In a more preferred

embodiment, cell conditioning solution comprises 0.4-8.2 mM sodium citrate; 1.8- 10 mM citric acid; 0.1-1% cell conditioning preservative; and 0.05-5% cell conditioning detergent. In a most preferred embodiment, cell conditioning solution comprises 8.2 mM sodium citrate; 1.8 mM citric acid; 0.05% cell conditioning preservative; and 0.1% cell conditioning detergent.

Complementary: The natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity may exist when only some of the nucleic acids bind, or when total complementarity exists between the nucleic acids.

Computer-readable media: Computer readable media or CRM refers to any device or system (e.g., machine or tool) for storing and providing information (e.g., instructions, etc.) to a computer processor. Examples of computer-readable media include, but are not limited to, a storage disk, a floppy disk, RAM, ROM, computer chips, digital video disc (DVDs), compact discs (CDs), hard disk drives (HDD), flash memory, and magnetic tape. A computer processor or central processing unit (CPU) are used interchangeably and refer to any hardware and software combination device that is able to read computer readable-media and perform a set of steps according to a program. An exemplary processor is a programmable digital microprocessor such as that available in an instrument that is used in performing automated staining of tissue samples as described herein, or it may be a microprocessor as found in a mainframe computer, a server, or a personal computer. For example, one or more steps or processes as exemplified in FIG. 7 for staining tissue for subsequent microscopic examination are provided by one or more tangible computer-readable media comprising instructions for performing the one or more steps or processes for automated methods for staining a tissue sample for microscopic examination.

Conjugating, joining, bonding or linking: Covalently linking one molecule to another molecule to make a larger molecule. For example, making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a hapten or other molecule to a polypeptide, such as an scFv antibody. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule ("EM"). The linkage can be either by chemical or recombinant means. "Chemical means" refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule. Coupled: When applied to a first atom or molecule being "coupled" to a second atom or molecule can be both directly coupled and indirectly coupled. A secondary antibody provides an example of indirect coupling. One specific example of indirect coupling is a rabbit anti-hapten primary antibody that is bound by a mouse anti-rabbit IgG antibody, that is in turn bound by a goat anti-mouse IgG antibody, which is covalently linked to a detectable moiety.

Detectable Moiety: A detectable compound or composition that is attached directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymes, haptens, and radioactive isotopes.

Epitope: An antigenic determinant. These are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope.

Hapten: A molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being immunogenic except in combination with a carrier molecule.

Homology: As used herein, "homology" refers to a degree of

complementarity. Partial homology or complete homology can exist. Partial homology involves a nucleic acid sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid.

Humanized antibody: An antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.

Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Pat. No. 5,585,089).

Immune Response: A response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. An immune response can include any cell of the body involved in a host defense response for example, an epithelial cell that secretes interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or

inflammation.

Immunogenic Conjugate or Composition: A term used herein to mean a composition useful for stimulating or eliciting a specific immune response (or immunogenic response) in a vertebrate. In some embodiments, the immunogenic response is protective or provides protective immunity, in that it enables the vertebrate animal to better resist infection or disease progression from the organism against which the immunogenic composition is directed. One specific example of a type of immunogenic composition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, under appropriate conditions, of stimulating the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal.

Mammal: This term includes both human and non-human mammals.

Similarly, the term "subject" includes both human and veterinary subjects.

miRNA, microRNA, miR: A non-coding RNA, typically between 18 and 25 nucleotides in length, which bind to complementary sequences in the 3 ' untranslated region of a target mRNA. Examples include, but are not limited to: let 7, let 7a, let 7a- 1, let 7b, let 7b- 1, let-7c, let-7d, let 7g, miR-1, miR-l-d, miR-1- 2, miR-7, (hsa-miR-7-1 - hsa-miR-7-3), (has-miR-9-1 - hsa-miR-9-3), miR-9, miR-lOa, miR-lOb, miR-15, miR-15a, miR-15b, miR-16, miR-16-1, miR-16-2, miR-17, miR-17-3p, miR-18a, miR-18b, miR-19a, miR19b-l, miR19b-2, miR-20a, miR-20b, miR-21, miR-22, miR-23, miR-23a, miR-23b, miR-24, miR-25, miR-

26a, miR-27a, miR-27b, miR-28, miR-29a, miR-29b, miR-29c, miR-30a-3p, miR- 30a, miR-30b, miR-30c, miR-30e-5p, miR-31, miR-32, miR-33, miR-34a, miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a, miR-100, miR-101, miR-103, miR- 105, miR-106b, miR-107, miR-108, miR-122, miR-124, miR-125, miR-125b, miR- 126, miR-127, miR-128, miR-129, miR-130, miR-130a, miR-132, miR-133, miR-

133a, miR-133a-2, miR-133b, miR-134, miR-135, miR-136, miR-137, miR-138, miR-139, miR-140, miR-141, miR-142, miR-143, miR-144, miR-145, miR-146a, miR-147, miR-148a, miR-149, miR-150, miR-151, miR-152, miR-153, miR-154, miR-155, miR-181a, miR-182, miR-183, miR-184, miR-185, miR-186, miR-187, miR-188, miR-190, miR-191, miR-192, miR-193, miR-194, miR-195, miR-196a, miR-197, miR-198, miR-199, miR-199a-l, miR-200b, miR-200c, miR-201, miR- 203, miR-204, miR-205, miR-206, miR-207, miR-208, miR-210, miR-211, miR-

212, miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-219, miR- 221, miR-222, miR-223, miR-224, miR-291-3p, miR-292, miR-292-3p, miR-293, miR-294, miR-295, miR-296, miR-297, miR-298, miR-299, miR-300, miR-301, miR-320, miR-321, miR-322, miR-323, miR-324, miR-325, miR-326, miR-328, miR-329, miR-330, miR-331, miR-333, miR-335, miR-337, miR-338, miR-339, miR-340, miR-341, miR-342, miR-344, miR-345, miR-346, miR-350, miR-361, miR-362, miR-363, miR-365, miR-367, miR-368, miR-369, miR-370, miR-371, miR-373, miR-380-3p, miR-409, miR-410, or miR-412, or functional variants thereof. Additional examples of known miRNAs are provided by U.S. Patent No. 8,003,320, which is incorporated herein by reference. Additional examples of human miRNAs can be found at: http://www.mirbase.org/cgi- bin/mirna_summary.pl?org=hsa, the contents of which are incorporated herein by reference.

Molecule of interest or Target: A molecule for which the presence, location and/or concentration is to be determined. Examples of molecules of interest include proteins, nucleic acid sequences, and miRNA tagged with a probe, such as a labeled or unlabeled locked nucleic acid probe.

Monoclonal antibody: An antibody produced by a single clone of B- lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

Multiplex, -ed, -ing: Embodiments of the present invention allow multiple targets in a sample to be detected substantially simultaneously, or sequentially, as desired, using plural different conjugates. Multiplexing can include identifying and/or quantifying nucleic acids generally, DNA, RNA, miRNA, peptides, proteins, both individually and in any and all combinations. Multiplexing also can include detecting two or more of a gene, a miR A, a messenger, and a protein in a cell in its anatomic context.

Neoplasia and Tumor: The process of abnormal and uncontrolled growth of cells. Neoplasia is one example of a proliferative disorder.

Neoplasm: The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma

(indolent and high grade forms), multiple myeloma, Waldenstrom's

macroglobulinemia, heavy chain disease, myelodysplasia syndrome, hairy cell leukemia and myelodysplasia. Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,

oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma). Protein: A molecule, particularly a polypeptide, comprised of amino acids. Examples include, but are not limited to: Caspase-3, FiNRK, p53 -related Tap63, PDCD4, PTEN, RECK, TPM1, ρ27^Μρ1, EGFR, Notch- 1, c-MET; Bcl-2, CdK4, Sirtl, Bmi-1, ZEB1, ZEB2, K-Ras, HMGA2, Cyclin Dl, Cyclin D2, Cyclin El, WNT3A, Pim-1, FoxPl, HDAC4, IGF1R, cyclin Gl, Erk5, IRS-1, Her2, Her3,

BMPRl, HuR, VEGF, FZD3, ITGA5, MMP16, RDX, RHoA, ΝΡκβΙ, EZH2, and ROCK1.

Sample, Sample material, material sample: A biological specimen containing genomic DNA, RNA (including mRNA and miRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. In one example, a sample includes a biopsy of an adenocarcinoma, a sample of noncancerous tissue, or a sample of normal tissue (from a subject not afflicted with a known disease or disorder). The term sample may be used to describe a biological specimen, as above, that is or is not present on a slide.

Specific binding moiety: A member of a specific-binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 10³ M^"1 greater,

10⁴ M^_1greater or 10⁵ M^"1 greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample).

Particular examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A), nucleic acids sequences, and protein-nucleic acids. Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins.

Stringency: A term used in hybridization experiments to denote the degree of homology between a probe and a target, such as a nucleic acid. In particular disclosed embodiments, a high stringency will indicate a high homology between the probe and the target. Substantially complementary: As used herein, "substantially

complementary refers to the oligonucleotides of the disclosed methods that are at least about 50% homologous to target nucleic acid sequence they are designed to detect, more preferably at least about 60%, more preferably at least about 70%>, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 90%, more preferably at least about 95%, most preferably at least about 99%.

Tissue Sample: Any sample comprising a component having a cellular organizational level between that of a cell and a complete organism, and may be selected from any tissue capable of biological analysis.

Tyr amide Signal Amplification: A method for amplifying chromogenic and fluorescent signals in immunohistochemistry protocols using a detectable moiety-labeled tyramine in combination with an enzyme, such as an oxido- reductase enzyme (e.g. horseradish peroxidase).

III. Automated Tissue Staining Systems and Methods

Automated systems typically are at least partially, if not substantially entirely, under computer control. Because automated systems typically are at least partially computer controlled, certain embodiments of the present invention also concern one or more tangible computer-readable media that stores computer- executable instructions for causing a computer to perform disclosed embodiments of the method. Particular disclosed embodiments concern a computer-controlled, bar code driven, staining instrument that automatically applies chemical and biological reagents to samples, such as tissue and/or cell samples, that are mounted or affixed to a slide. More than one slide may be used, with particular

embodiments using from about 1 to about 50 slides; more typically from about 1 to about 20 slides.

The present invention can be used with any of various automated staining systems, particularly those provided by Ventana Medical Systems, Inc., including the Benchmark XT, Benchmark Ultra, and Discovery systems. Exemplary systems are disclosed in U.S. Patent No. 6,352,861, U.S. Patent No. 5,654,200, U.S. Patent

No. 6,582,962, U.S. Patent No. 6,296,809, and U.S. Patent No. 5,595,707, all of which are incorporated herein by reference. The following description exemplifies a suitable embodiment of an automated method and system. Additional information concerning automated systems and methods also can be found in PCT/US2009/067042, which is incorporated herein by reference.

Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed.

Embodiments of automated systems may perform all or any subset of, processing steps of processing, staining and coverslipping of slide-mounted biological samples. Samples on slides are conducted through a sequence of steps including deparaffinizing the tissue sample by contacting it with deparaffinizing fluid at a temperature above the melting point of the paraffin; rinsing liquefied paraffin away; staining the tissue sample by contacting it with a staining reagent, or staining reagents sequentially or simultaneously, depending on a particular desired protocol; and coverslipping the slide.

A thin slice or section of sample material can be disposed on a first surface of the slide. The material can be, for example, a tissue sample which has been appropriately prepared for receiving the fluid treatments described herein. "Sample material" or "material sample" refer to any material that can be disposed and treated on a slide for analysis, including any tissue or biological sample obtained from, derived from, or containing any organism including a plant, an animal, a microbe, or even a virus. Particular examples of such sample materials can include a collection of cells, such as sections of organs, tumors sections, bodily fluids, smears, frozen sections, cytology preps, and cell lines. As described in U.S.

Publication No. 2005/0164374 (incorporated herein by reference), before being disposed on the slide, the sample material can be frozen or fixed, dehydrated, treated with a wax or other plastic substance, further sliced, and/or exposed to other solvents or treatments. The type and size of the sample material, along with its prior treatment, can vary depending on the particular analysis being performed.

One embodiment of a suitable apparatus comprises a station base that has a side surface, a slide conveying device, and a fluid dispensing device. The slide conveying device is configured to receive a microscope slide having a material sample disposed on a first surface of the microscope slide. The slide conveying device is further configured to move the microscope slide from a first position to a second position, with the microscope slide being in a substantially vertical orientation when the microscope slide is in the second position. The fluid dispensing device is configured to dispense a volume of fluid while the microscope slide is in the first position. At least a portion of the dispensed fluid contacts one or more of the first surface of the microscope slide.

In specific implementations, the volume of fluid dispensed by the fluid dispensing device is between about 0 and 100 microliters, and, more specifically, between about 0 and 30 microliters and between about 30 and 100 microliters. In other specific implementations, the slide conveying mechanism is configured to receive and move a plurality of slides. In addition, the side surface of the station base can be configured to treat a plurality of slides at one time.

In another embodiment, an automated apparatus for treating a plurality of slides with a thin film of fluid is provided. The apparatus comprises a treatment station, a slide conveying device, and a fluid dispensing device. The treatment station comprises a station base that has a side surface. The slide conveying device is configured to receive a plurality of microscope slides. Each microscope slide has a material sample disposed on a first surface of each respective microscope slide. The slide conveying device is configured to carry the plurality of microscope slides and move the microscope slides to and from the treatment station. A fluid dispensing device is provided at the treatment station. When one of the plurality of slides is positioned at the treatment station, the fluid dispensing device dispenses a desired fluid volume.

In specific implementations, the slide conveying device is configured to independently move each of the plurality of microscope slides to and from one or more treatment stations. In other specific implementations, the apparatus further comprises a separate incubation housing and the slide conveying mechanism is configured to move the microscope slides from the treatment station to the incubation housing.

In another embodiment, a method of treating a microscope slide with a thin film of fluid is provided. The method comprises providing a microscope slide with a material sample disposed on a first surface of the microscope slide, providing a station base having a side surface, and providing a fluid dispensing device. The microscope slide is positioned with the first surface of the microscope slide facing the side surface of the station base. A volume of fluid is dispensed from the fluid dispensing device so that at least a portion of the dispensed fluid contacts one or more of the first surface of the microscope slide and the side surface of the station base.

Disclosed embodiments typically include a deparaffinizing station.

Deparaffinization can be accomplished using any suitable protocol. Solely by way of example, embodiments of a method for deparaffinizing are described in U.S.

Patent No. 6,855,559, assigned to Ventana Medical Systems, Inc., and incorporated herein by reference. Briefly, a paraffin-embedded biological sample on a glass microscope slide is heated using a heating element. Heating the sample can be used to accomplish various goals, such as to melt the inert material, including paraffin and/or to drive off any water which may be between the paraffin section and the glass to allow the charge of the tissue to adhere to the glass. The inert material may be removed from the slide by a applying to the sample a fluid suitable to dissolve the inert material. Reagents can be used instead of or in addition to heating the embedded biological samples. Suitable reagents include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil, and PAG oil. Each of these reagents also may contain ionic or non- ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate.

Various deparaffinizing agents may be used, and preferably comprise aqueous-based fluid such as disclosed in co-pending U.S. patent application Ser. No. 09/721,096 filed Nov. 22, 2000 and U.S. Pat. No. 6,544,798, issued Apr. 8, 2003, including deionized water, citrate buffer (pH 6.0-8.0), tris-HCl buffer (pH 6- 10), phosphate buffer (pH 6.0-8.0), FSC buffer, APK wash™, acidic buffers or solutions (pH 1-6.9) basic buffers or solutions (pH 7.1-14), which are given as exemplary. If desired, the aqueous-based fluid may also contain one or more ionic or non-ionic surfactants such as Triton X-100™, Tween™, Brij, Saponin and Sodium Dodecylsulfate. Typically, the deparaffinizing fluid is heated. For example, if the embedding medium is paraffin, which has a melting point between 50-57 °C, the fluid should be heated to a temperature greater than the melting point of paraffin, e.g. between 60-70 °C. This thermal platform eliminates the need to use harsh chemicals (e.g. xylenes) for deparaffinization. Typically, the fluid is heated in the fluid supply.

Following the deparaffinization step, a deparaffinization rinse step may be performed. This step usually is performed using a lower alkyl (10 carbon atoms or fewer) alcohol, such as methanol, ethanol and/or isopropanol. For disclosed embodiments, this rinse step typically involved applying 4 milliliters of the selected alcohol to each slide.

Following the deparaffinization step, slides move into position for receiving a fluid treatment. A volume of fluid is desirably dispensed from a fluid dispensing device (e.g., a nozzle). A fluid dispensing device is desirably connected to a reservoir that contains one or more reagents or other fluids, with the fluid dispensing device being positioned and/or oriented so that at least a portion of the dispensed volume of fluid enters a wetting region. The "wetting region" is defined herein as the region at or adjacent to the area where the slide contacts or abuts the base (or membrane).

Fluids can be dispensed onto the slide at a position above the wetting region and the fluid can flow downward into the wetting region. The volume of fluid can be "painted," "misted," ink-jetted, and/or dispensed onto the wetting region or onto the slide surface in any manner effective to achieve the desired result(s), including wetting the sample.

The dispensing of reagents and other fluids is desirably automated. Thus, the selection of the fluid to be dispensed and the amount and manner in which the fluid is dispensed can be computer controlled. U.S. Patent Publication

2008/0102006, the entire disclosure of which is incorporated herein by reference, describes robotic fluid dispensers that are operated and controlled by

microprocessors.

Particular applications require that the material be incubated for a specified amount of time with the fluid in contact with the material. The slide can remain in the position for the length of time required, if desired.

Various fluids, typically aqueous solutions, are used with the system.

Examples of aqueous fluid include de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK

Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1- 14), mineral oil, Norpar, canola oil, and PAG oil. Moreover, the aqueous fluid may also contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate. The surfactants lower the surface tension of the aqueous fluid, allowing the aqueous fluid to spread better over the surface of the slide. In particular embodiments, the aqueous fluid may include de -ionized water with about 0.1% Triton X-100. An additional ingredient may be added, acting as an anti-microbial agent, so that the fluid prior to application on the slide does not contain microbes. In other embodiments, the fluid may include a water content, by weight, of 99% or greater (i.e., the fluid is composed of between 99%- 100% water). Using water as a fluid to remove the embedding material is unlike what is conventionally used to remove the embedding material, such as organic solvents.

Further, the aqueous fluid should be applied in sufficient amounts and at sufficient times (accounting for evaporation of the aqueous fluid due to heating) such that the embedding media may float to the surface of the aqueous fluid and such that the biological sample on the slide will not dry out. In some embodiments, the aqueous fluid may be applied sequentially, with a first application of approximately 1 mL of aqueous fluid on the biological sample, and with a second application two minutes later of aqueous fluid. The second application may be approximately 0.5 mL to 1 mL of aqueous fluid. The fluid may be applied to the slide by using a nozzle which is positioned directly above the slide. In this manner, the amount of fluid dropped onto the slide may be controlled. Moreover, because the fluid embedding material (such as paraffin) may have a low surface tension, applying a stream of fluid onto the slide may not leave a sufficient amount of fluid on the top of the slide. Thus, using the nozzle to drop the fluid onto the embedded sample is preferred as it allows more of the fluid to remain on the upper surface of the slide

The reagents used for cell conditioning can be the same as those for exposing the embedded biological sample. For example, for DNA targets, a cell conditioning solution may be a solution of saline sodium citrate (SSC); a common temperature setting may be 95 °C for a duration ranging from 2-90 minutes. For protein targets, a cell conditioning solution may be a solution of phosphate buffer; a common temperature setting may be in excess of 100° C for a duration ranging from 2-90 minutes. For RNA targets, a cell conditioning solution may be a solution of SSC; a common temperature setting may be 75 °C for a duration ranging from 2-90 minutes.

The terms "Reagent", "Buffer", "Additive", "Component" and "Solution" as used herein for exposing or deparaffinizing (i.e., the process of deparaffinization) may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: de-ionized water, de -ionized water with about 0.1% Triton X- 100, 10 mM phosphate at around pH 6.1 , 10 mM phosphate with about 0.1% Triton X-100 at around pH 6.1, 10 mM citrate at around pH 6, 10 mM citrate with about 0.1% Triton X-100, 0.3 M sodium chloride and 30 mM trisodium citrate (hereinafter referred to as 2xSSC, 10 mM

Tris[hydroxymethyl]aminomethane chloride (i.e., Tris-Cl) at around pH 8.2, 10 mM Tris-Cl with about 0.1% Triton X-100 at around pH 8.2. A person having ordinary skill in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for exposing or deparaffinizing.

The terms "Reagent", "Buffer", "Additive", "Component", "Solution" and

"Cell Conditioner" as used herein for cell conditioning may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: 5 mM citrate at around pH 6, 5 mM citrate with about 0.5% sodium dodecyl sulfate (SDS) at around pH 6, 10 mM citrate at around pH 6, 10 mM citrate with about 0.5% SDS at around pH 6, 20 mM citrate at around pH 6, 20 mM citrate with about 0.5% SDS at around pH 6, 50 mM citrate at around pH 6, 50 mM citrate with about 0.5% SDS at around pH 6, 1 mM ethylene diamine tetraacetic acid (EDTA) at around pH 8, 1 mM EDTA with about 0.075% SDS at around pH 8, 10 mM EDTA at around pH 8, 10 mM EDTA with about 0.075% SDS at around pH 8, 20 mM EDTA at around pH 8, 20 mM EDTA with about 0.075% SDS at around pH 8, 50 mM EDTA at around pH 8, 50 mM EDTA with about 0.075% SDS at around pH 8, 10 mM citrate with about 0.5% SDS and about

1% ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about 5% ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about 10% ethylene glycol at around pH 6, 1 mM EDTA with about 0.075% SDS and about 1% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS and about 5% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075%

SDS and about 10% ethylene glycol at around pH 8, phosphate/citrate/EDTA at about pH 9, 10 mM citrate with about 10 mM urea at around pH 6, 10 mM citrate with about 1 mM urea at around pH 6.2, 10 mM sodium citrate with about 1.4 mM MgCl₂ and about 0.1% SDS at around pH 7, 10 mM sodium citrate with about 1.4 mM MgCl₂ and about 0.1% SDS at around pH 7.99, 10 mM Tris-Cl at around pH 8, 10 mM Tris-Cl with about 20% formamide at around pH 8, 10 mM citrate with about 5% dimethyl sulfoxide (DMSO) at around pH 6, 10 mM citrate with about 0.1% Triton X-100 and about 20% formamide at around pH 6, 10 mM phosphate with 5xSSC and about 2.5% chrondroitin A at around pH 7, 10 mM Tris-Cl with about 10 mM EDTA and about 0.1% Triton X- 100 and about 20% formamide at around pH 8.2, 10 mM citrate with about 20% glycerol at around pH 6, 10 mM citrate with about 0.1% Triton X-100 and about 10 mM glycine at around pH 6, 1 mM EDTA with about 1 mM citrate and about 0.25% SDS at around pH 7.8, Norpar/mineral oil (high temperature coverslip), PAG- 100 oil, 10 mM citrate with about 2% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 1%

SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.5% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.25% SDS at a pH of around 6 to around 6.2, 1 mM EDTA with about 2% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 1% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.5% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.25% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.1 % SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.075% SDS at a pH of around 7.5 to around 8, 0.5 mM EDTA with about 0.25% SDS at around pH 8, 10 mM EDTA with about 0.5% SDS at around pH 9.6. A person of ordinary skill in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for cell conditioning.

Automated mechanisms for transporting a plurality of slides can be used to locate the slides in a position for fluidic treatment. Each slide can be removably coupled to a slide clip, which is in turn removably coupled to a conveying device. Conveying devices can be configured to provide movement of slides in a variety of manners. Slide movement can be an indexing movement such that each individual slide is indexed to adjacent treatment positions. If necessary, the slide can be lowered or raised to the desired height for application of a fluidic treatment. For example, a stepper motor or other mechanism can be configured to move the slides to position them to receive fluid treatment. Multiple slides can be treated simultaneously, if desired, at different fluid treatment stations. In addition, a plurality of fluid treatments can be performed along the path of travel achieved by a single conveying device. Moreover, multiple conveying devices can be used and the slides can be transferred from one conveying device to another by any automated or manual mechanism.

Fluid treatments may be configured so that specific treatments occur at a specific "station." The number of slides that can be processed through any given system is controlled by the time it takes to perform operations (treatment) at each station, and the time for each treatment varies depending on the particular treatment being performed. Thus, new slides cannot be treated until the slides currently being treated are processed and moved away from the treatment station.

Conventional fluid processing stations are generally configured to treat a slide with a fluid and then maintain the slide at the same location while it is incubated. In illustrative embodiments, the automated instrument is devoid of any reagent baths in which the samples is submerged. Furthermore, in illustrative embodiments, the methods and processes described herein are devoid of steps in which the sample is submerged in a reagent bath. Manual or automated processes that include the use of reagent baths for treating samples are known to present patient safety risks. Substantial evidence has been amassed demonstrating that methods that include reusing reagent baths for histopathology samples may result in cross-contamination of samples which can lead to misdiagnosis. A percentage of samples exposed to reagents in a bath will lose adhesion to the substrate and remain in the bath after the substrate is removed.

As discussed above, after the slide is disengaged from the treatment station, a thin film of the applied fluid remains on the slide. Because the thin fluid film is desirably small (i.e., only a portion of the applied volume of fluid) and desirably relatively viscous, the slides can be transported and incubated at a location away from the fluid treatment station. Thus, after receiving a fluid treatment as described herein, slides can be transported and garaged during an incubation phase without concern for fluid spillage due to gravitational effects or the acceleration effects involved in moving the slides to the new location. Slides can move sequentially through the length of the incubation garages. The length of the garage as well as the speed at which the slides are moved can be selected to provide sufficient time for slide incubation. After being incubated, slides can be transported to another fluid application station and the process can be repeated. Alternatively, if desired or if the films are larger or less viscous, the slides can be treated with a fluid as described above and then oriented horizontally for transport to an incubation garage or other treatment, rinse, or storage location. The slides can undergo any of a variety of fluid or other treatments, either before or after being transported to the incubation garages. Thus, various sequential fluid application stations can be provided. For example, a reagent treatment can be performed at a first treatment station and a rinse treatment can be performed at a second treatment station. Alternatively, the reagent treatment and rinse treatment can be performed at a single treatment station. A plurality of fluid dispensing devices (such as described herein) can be configured to dispense one or more treatment fluids to the plurality of fluid treatment stations. The fluid treatment stations can be configured so that each slide undergoes the same fluid treatment at the same time or the fluid treatment stations can be configured so that each slide undergoes a fluid treatment that is different from the other slides. For example, a single slide can be positioned at a first fluid treatment station and be subjected to a first fluid treatment. After the first fluid treatment is completed, the slide can be moved to a second fluid treatment station where it can receive a second, different fluid treatment. In this manner, a single station base can be configured to provide a plurality of sequential fluid treatments to a single slide. A single slide can also be treated at a single base without moving the slide laterally to a new treatment position along the base. If desired, for example, the slide and base can be rinsed prior to performing a second fluid treatment at a single treatment location.

The automated system can include a heating and/or cooling device, such as a resistive heater, a radiant heater configured to heat one or more surfaces (such as the side surface) of the base station or a Peltier device coupled with or integral to the station base. Thus, for example, one or more slides can be treated at a station and then heated or treated at the same location. In addition, one or more stations can be enclosed in one or more covered or sealed chambers, within which one or more chambers the environment (such as humidity levels, temperature and/or pressure) can be independently or simultaneously controlled. Each station base also can be modular. Thus, for example, a single station base can be a modular element that is substantially enclosed with an independently controlled heating device. In this manner, the modular element can receive one or more slides and the entire modular element can be inserted into a larger unit or housing, such as a carousel or other such slide receiving apparatus. Each sample within the apparatus can receive an individualized staining and/or treatment protocol even when the protocols require different temperature parameters. In addition, the temperature of the entire slide can be carefully controlled (e.g. within +1-2 °C of the desired temperature) and the entire slide can be maintained at a constant temperature. This temperature control is useful in hybridization/denaturation steps, which are discussed herein. U.S. Patent Publication 2003/0211630, the entirety of which is incorporated herein by reference, describes examples of housings that can receive and dispense fluid onto

Software operates the system sequence and schedules the operations performed by the various functional workstations on each tray of microscope slides. The system can handle plural trays at one time with each tray requiring the operations performed by one or more workstation and perhaps multiple visits to the same workstation. This integrated automated system provides high throughput staining of biological samples on slides. Clean, fresh or constantly filtered de- paraffinizing agent, or staining reagent, is used to eliminate the possibility of cell carryover from slide to slide.

Automation provides consistency and reproducibility that manual methods cannot achieve. Previously described methods are slow, labor-intensive, and difficult to reproduce. Correspondingly, these methods carry a high expense in terms of labor and time. Automation has heretofore been impractical because of the multiple fixation steps that were believed to be necessary. Our inventive method uses distinct fixation processes. Illustratively, these distinct fixation processes are unique to automated instruments and provide the ability to automate the dual staining procedure. The automated processes also provide superior results than the manual methods with respect to patient safety, reproducibility, and efficiency.

Conventional wisdom has been that ISH staining procedures require enzymatic pretreatments. In particular, it has been theorized that adequate expression of nucleic acid targets using automated instrumentation requires protease treatments. Without being bound to a particular theory, protease is thought to "punches holes" in the proteins making up a tissue sample thereby enabling efficient sample penetration by nucleic acid probes. As such, routine ISH procedures, both automated and manual, use proteases for exposing nucleic acid targets. An example of such a reagent are ISH-Proteases (Ventana Medical Systems, Inc., Tucson, AZ; Catalog #s: 760-2018, 760-2019, 760-2020). One aspect of the present disclosure is that ISH proteases are disadvantageous to protein detection or when coupled with protein expression steps result in damaged tissue morphology. To overcome these long-felt obstacles, a non-enzymatic approach to target expression was discovered that conserves and permits the detection of both miR A and proteins so that a single sample can be analyzed for both

simultaneously if desired. The efficacy of this procedure was tested using various model tissues (e.g. lung, tonsil, and breast) with both scramble probes and U6 probes to verify penetration and specificity.

miRNA analysis has typically used PCR or northern blots, neither of which provide the tissue context important for diagnostics. In contrast to PCR and northern blots, methods according to the present disclosure offer the distinct advantage of providing contextual information about the sample (e.g. the ability to visualize the relative distribution of miRNA and proteins in a heterogeneous population of cells in a tissue). This information provides medical value beyond what was possible using solution-based detection approaches such as PCR. PCR also has various complicating factors (using completely RNAase free solutions) that are completely avoided by using the inventive method. The inventive method enables researchers to identify and validate hypothesized signaling pathways which would not be possible with prior art methods that do not preserve contextual information about the sample and the ability to visualize both the miRNA species and its target protein in the same sample.

IV. miRNA and Proteins

A person of ordinary skill in the art will appreciate that the present method can be used to detect any desired miRNA. "miRNA," "microRNA," or "miR" means a non-coding RNA, typically between 18 and 25 nucleobases in length, which bind to complementary sequences in the 3 ' untranslated region of a target mRNA. miRNAs are abundantly present in all human cells and each miRNA is able to repress multiple targets. The disclosed method and apparatus may be used to target particular proteins, such as proteins involved in cancer proliferation. In illustrative embodiments, the method includes the detection of the miRNA and protein on a single tissue sample so that the relationship between the tissue morphology, as observed through primary staining can be visualized concurrently with the stains associated with both the miRNA and the protein. Referring now to FIG. 1(A)-(C), shown are photomicrographs showing serial sections of a breast cancer tissue stained using a dual staining procedure, as described herein. FIG. 1(A) shows the dual detection of HER3 (detected with DAB) and miR-205

(detected with NBT/BCIP). The cellular morphology is visualized using primary hematoxylin and eosin (H&E) staining. FIG. 1(B) shows a control in which the dual staining procedure was done without miR-205 probe. This figure shows that the signal observed for the miR-205 in the dual-stain shown FIG. 1(A) is specific for the interaction of the miR-205 probe with the miR-205 in the sample.

Similarly, FIG. 1(C) shows a control in which the dual staining procedure was done without anti-HER3 demonstrating that the HER3 signal observed in FIG. 1(A) is specific anti-HER3 binding to the sample. Referring now to FIG. 2(A)-(B), shown are photomicrographs demonstrating exemplary results obtained using a dual stain protocol for miR-205 (detected with NBT/BCIP) and Bcl2 (detected with DAB) on (A) a first breast cancer tissue and (B) a second breast cancer tissue.

Referring to FIG. 3(A)-(D), shown are photomicrographs of serial sections of a lung tissue stained using a dual staining procedure, as described herein.

FIG.3(A) shows the dual detection of CRK (detected with DAB) and miR-126 (detected with NBT/BCIP). As described above with respect to HER3 and miR- 205, FIG. 3(B)-(C) show controls which demonstrate the specificity of the detection for the miR-126 probe and the anti-CR . FIG. 3(D) shows a control in which the dual staining procedure was done without anti-CRK and using a scramble probe in place of the miR-126 probe. This control was done to further demonstrate the specificity of the miR-126 probe.

Particular proteins that may be targeted include oncogenes and other proteins, such as HER, Ras, Myc, ANP32A, SMARCA4, COL3A1 (a gene up- regulated in advanced carcinoma), Gabl (which is involved in cell proliferation),

ING4 (a homo log of tumor suppressor p33, which stimulates cell cycle arrest, repair and apoptosis), LASS2 (a tumor metastasis suppressor), and CNOY7 (a gene expressed in colorectal cell lines and primary tumors).

V. Specific Binding Moieties, Detectable Moieties, and Detection Systems

The present invention is particularly directed to a method and automated system for dual detection of miRNA and protein or proteins associated with regulating the miRNA. A person of ordinary skill in the art will appreciate that any suitable specific binding moiety and detectable moiety suitable for detecting miRNA and proteins by contacting a sample with such reagent or reagents now known or hereafter discovered can be used to practice disclosed embodiments. Particular exemplary specific binding moieties, detectable moieties, and detection systems are disclosed below.

A. Locked Nucleic Acid probes

Locked nucleic acid (LNA) is a modified RNA nucleotide comprising a methylene bridge that locks the ribose in a particular conformation, which causes enhanced base stacking and backbone preorganization. In particular disclosed embodiments, the LNA can be hybridized with the miRNA in order to increase the ability of the miRNA to bind to the probe. In particular, the tendency of the miRNA to dissociate from the target is increased at temperatures commonly used in in-situ analysis because the complex tends to have a low melting temperature (T_m). This tendency to dissociate can be attributed to the miRNA's small size. The T_m of the complex can be increased by hybridizing the miRNA with one or more LNA compounds. In particular disclosed embodiments, the T_m of the

miRNA/LNA complex may range from about 50 °C to about 90 °C; more typically from about 70 °C to about 85 °C. Because the LNA comprises a locked ribose ring, the ability of the hybridized probe to move is limited, and therefore the probe is better able to associate, or bind, with the target protein. The miRNA/LNA complex can be used for different samples, such as cells, tissues, whole mounts, FFPE samples, and frozen samples.

Particular disclosed embodiments concern probes comprising LNAs that are positioned to achieve high sequence specificity, low secondary structure, and minimal self-annealing. In particular disclosed embodiments, the probe may be labeled, such as with a detectable moiety (e.g. a hapten and/or a fluorophore) or unlabeled. Exemplary probes include, but are not limited to, miRCURY LNA™ probes for has-miR-205, oan-miR-205, cin-miR-126, and dre-miR-126, which may be labeled or unlabeled.

B. Haptens as Detectable Moieties

Various suitable haptens can be used to practice disclosed embodiments of the present invention. These include haptens conventionally used for processing tissue samples, including digoxigenin, dinitrophenyl, biotin, fluorescein, rhodamine, bromodeoxyuridine, mouse immunoglobulin, or combinations thereof. Other haptens currently being developed also can be used, including an oxazole, a pyrazole, a thiazole, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azoaryl or a benzodiazapine.

Additional information concerning these haptens is provided by U.S. Patent No. 7,695,929, entitled Haptens, Hapten Conjugates, Compositions thereof, and Method for their Preparation and Use, which is hereby incorporated by reference herein in its entirety. Plural haptens may be coupled to a polymeric carrier. See, U.S. Patent No. 7,985,557, entitled Polymeric Carriers for Immunohistochemistry and in situ Hybridization, which is incorporated herein by reference, for additional information concerning polymeric carriers. Haptens can be detected using specific binding pairs. For example, biotin can be detected using streptavidin. Haptens also can be detected using anti-hapten antibodies. For example, digoxigenin can be detected with an anti-digoxigenin antibody, coupled to another detectable moiety, such as a fluorophore or enzyme.

VI. Process Steps and Reagents used for Dual Detection of miRNA and Protein Targets

Embodiments of a fully automated method for dual staining and detection of miRNA and protein are disclosed. A miRNA target and a protein target, such as a protein that may be regulated by the miRNA, are stained and detected in a single sample mounted on a slide, e.g., cells, tissues, whole mounts, formalin- fixed, paraffin-embedded (FFPE) samples, or frozen samples. Embodiments of the method include miRNA probe hybridization, miRNA probe detection, and protein detection. In some embodiments, antigen retrieval (e.g., cell conditioning) is performed prior to miRNA probe hybridization.

A. Deparafflnization

If the sample is an FFPE tissue section, deparafflnization is performed before staining and detecting the miRNA and protein targets. In some

embodiments, deparafflnization is performed by heating a slide containing the sample to a temperature sufficient to melt the paraffin and/or to drive off any water in the sample. Typically, the slide is heated to 70-80 °C for a period of time, such as for about 2-10 minutes. A deparaffinizing reagent also may be added to the slide to dissolve the paraffin. The slide then is rinsed to remove dissolved paraffin and the deparaffinizing reagent. In some embodiments, a plurality of deparaffinization cycles, such as from one to about five cycles, is conducted. In a working embodiment, three deparaffinization cycles were performed at 65 °C for 4 minutes.

B. Antigen Retrieval

Certain disclosed embodiments concern an antigen retrieval step. Antigen retrieval is variously referred to as epitope retrieval, antigen retrieval, cell conditioning, target retrieval, or target expression or like terms known in the art. One aspect of the present disclosure is that target retrieval for proteins and target retrieval for nucleic acid targets have historically been divergent methodologies. Developing a procedure that maintains tissue morphology, is compatible with tissue adhesion on the slide, and which adequately results in the expression of both miRNA targets and protein targets was a challenge overcome within the scope of the present discovery. In illustrative embodiments, non-enzymatic cell conditioning will be used wherein no protease is required. Cell conditioning may involve any number of the following steps and the steps may occur in any order. The following description is meant to be illustrative and not limiting in any way. In particular disclosed embodiments, the sample may be first treated with an amount of a cell conditioning fluid at a reaction temperature ranging from about 80 °C to about 95 °C; more typically from about 90 °C to about 93 °C. In certain disclosed embodiments, the sample may be exposed to the cell conditioning fluid in a number of cycles, such as 1 to about 5 cycles. The amount of time during which the sample is exposed to the cell conditioning fluid may vary. In particular disclosed embodiments, the sample is exposed to the cell conditioning fluid for about 2 minutes to about 10 minutes; more typically for about 4 minutes to about 8 minutes. In a working embodiment, the reaction proceeded for 8 minutes.

The cell conditioning fluid may be selected from any fluid having one or more of the following characteristics: (a) high boiling point; (b) low vapor pressure; (c) stability at reaction temperatures; (d) fluidically stable; and (e) low viscosity. In particular disclosed embodiments, the cell conditioning fluid may be a buffer having a slightly basic pH. For example, a slightly basic pH comprises a pH at which the buffer is capable of disrupting covalent bonds formed by formalin in tissue at elevated temperatures. Exemplary embodiments concern using a Tris- based buffer having a pH ranging from about 7.7 to about 9; more typically, the Tris-based buffer has a pH of 8.0.

Proteinase K has historically been used for antigen retrieval from tissue samples prior to miRNA ISH. For example, an antigen retrieval step may have historically included treating a tissue sample with a 10-15μg/mL solution of proteinase K. One aspect of the present invention is that it was discovered that proteinase K adversely affected subsequent protein staining and tissue morphology in connection with an automated staining procedure. Referring now to FIGS. 4(A)- (B), shown are images illustrating the deleterious effects associated with proteinase conditioning on a sample used to detect HER3. The sample shown in FIG. 4(A) is a breast cancer specimen stained according to methods disclosed herein except that an enzymatic antigen retrieval step (proteinase K) was used. Conversely, FIG. 4(B) shows the breast cancer specimen prepared using a non-enzymatic antigen retrieval step. It was determined that substantial beneficial results were obtained using a cell conditioning process comprising using of a buffer and heat

preconditioning instead of proteinase pretreatment for detecting HER3. FIG. 5(A) is an image illustrating the deleterious effects associated with detecting miRNA within a dual staining protocol when using proteinase conditioning for dual miRNA and protein detection of a sample comprising miR-205. FIG. 5(B) is an image illustrating the substantial beneficial difference obtained by using a buffer and heat preconditioning instead of proteinase pretreatment for dual detection of a sample comprising miR-205. One aspect of the present disclosure is that the dual staining procedure places an additional burden on the tissue sample's integrity such that even miRNA singly stained tissue is not presentable when the proteinase pretreatment is used. This is unexpected in light of conventional wisdom that would indicate that a singly stained miRNA sample would be presentably stained subsequent protease target retrieval.

Following cell conditioning, the sample is rinsed with a reaction buffer to remove the cell conditioning fluid and/or prepare the sample for subsequent target detection. In particular disclosed embodiments, the reaction buffer may comprise Tris(hydroxymethyl)aminomethane, acetic acid, and a preservative solution

(PROCLIN® 300, SAFC Supply Solutions, Saint Louis, MO). After being treated with the reaction buffer, the sample may be rinsed with a solution capable of controlling stringency for subsequent steps. In particular disclosed embodiments, the solution may be an acid salt buffer solution, such as saline-sodium citrate (SSC).

C. Probe Hybridization

Following antigen retrieval, the targets are specifically labelled. The miRNA and protein targets may be labelled simultaneously or sequentially in any order. However, in certain embodiments, superior miRNA detection is achieved when miRNA labeling and detection is performed prior to protein labeling and detection.

Detecting the miRNA target includes contacting the sample with a nucleic acid specific binding moiety capable of recognizing and binding to a particular miRNA target, and subsequently detecting the nucleic acid specific binding moiety. In some embodiments, the nucleic acid specific binding moiety is a locked nucleic acid probe capable of hybridizing to the miRNA target. The nucleic acid specific binding moiety may be conjugated to one or more detectable moieties, such as an enzyme, a fluorophore, a luminophore, a hapten, a fluorescent nanoparticle, or a combination thereof. In some embodiments, the detectable moiety is a hapten selected from an oxazole, a pyrazole, a thiazole, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azoaryl, or a benzodiazepine. In certain embodiments, the hapten is digoxigenin, dinitrophenyl, biotin, fluorescein, rhodamine, bromodeoxyuridine, mouse immunoglobulin, or a combination thereof. In one embodiment, the nucleic acid specific binding moiety comprises a locked nucleic acid probe coupled to a polymeric carrier to which a plurality of haptens is coupled.

The sample is contacted with the nucleic acid specific binding moiety, or probe, under conditions sufficient to enable recognition and binding of the nucleic acid specific binding moiety to the miRNA target. In illustrative embodiments, about 500 fmol to about 1 ,000 fmol of probe is used. In working embodiments, about 700-800 fmol, such as about 750 fmol, of probe was used. The probe and sample are incubated in a hybridization buffer at a first temperature and for a first period of time sufficient to denature the sample. For example, the probe and sample may be incubated for a time period ranging from about 5 minutes to about 15 minutes at a temperature ranging from about 70 °C to about 90 °C. The probe and sample then are incubated for a second period of time at a second temperature to facilitate hybridization of the probe to the target miRNA. The second temperature typically is about 30 °C below the melting temperature (T_m) of the probe. In some embodiments, the hybridization temperature ranges from about 50 °C to about 65 °C. The second period of time is sufficient to allow probe hybridization. In certain embodiments, the second period of time is about one hour. Following hybridization, the slide is washed (e.g., with saline-sodium citrate buffer) to remove any unbound and/or non-specifically bound probe.

In some embodiments, the nucleic acid specific binding moiety is a locked nucleic acid probe coupled to a detectable moiety, and detecting the miRNA target further comprises detecting the detectable moiety. The detectable moiety may be detected by adding an anti-label antibody to the slide. For example, a hapten- labeled probe may be detected with an anti-hapten antibody, such as a mouse anti- hapten antibody. In a working embodiment, a digoxigenin-labeled locked nucleic acid probe was used to bind to the miRNA, and about 200 ng of a mouse anti-DIG antibody was used to detect the probe.

D. Detection

In certain embodiments, components of a first detection system then are added to the slide to detect the anti-label antibody. In some examples, the first detection system comprises an enzyme conjugated to a secondary antibody (e.g., an anti-mouse antibody), an enzyme substrate, and optionally one or more additional components. In a working embodiment, the first detection system included an anti- mouse antibody-alkaline phosphatase conjugate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and nitro-blue tetrazolium chloride (NBT). The anti-mouse antibody-alkaline phosphatase conjugate is added to the slide, and binds to the anti- label antibody. In a working embodiment, the slide was incubated with anti-mouse antibody-alkaline phosphatase conjugate for 16 minutes. The slide then is washed to remove unbound antibody-enzyme conjugate. BCIP and NBT then are added to the slide. Alkaline phosphatase cleaves the phosphate of the BCIP, which reduces NBT to produce a detectable blue color. In some embodiments, a plurality of incubation cycles with the first detection system may be performed. In a working embodiment, two incubation cycles of 44 minutes with BCIP and NBT were performed.

Both IHC and ISH involve a specific recognition event between a nucleic acid probe (ISH) or an antibody (IHC) and a target within the sample. This specific interaction labels the target. The label can be directly visualized (direct labeling) or indirectly observed using additional detection chemistries. Chromogenic detection, which involves the deposition of a chromogenic substance in the vicinity of the label, involves further detection steps to amplify the intensity of the signal to facilitate visualization. Visualization of the amplified signal (e.g. the use of reporter molecules) allows an observer to localize targets in the sample.

In an illustrative embodiment, the method includes chromogenic detection. Chromogenic detection offers a simple and cost-effective method of detection. Chromogenic substrates have traditionally functioned by precipitating when acted on by the appropriate enzyme. That is, the traditional chromogenic substance is converted from a soluble reagent into an insoluble, colored precipitate upon contacting the enzyme. The resulting colored precipitate requires no special equipment for processing or visualizing. Table 1 is a non-exhaustive list of chromogen systems useful within the scope of the present disclosure: Table 1: Chromogenic detection reagents.

Table 1 , while not exhaustive, provides insight into the varieties of presently available chromogenic substances (†WO2012/024185, Kelly et al. "Substrates for Chromogenic detection and methods of use in detection assays and kits").

Detecting the protein target includes contacting the sample with a protein specific binding moiety under conditions sufficient to enable recognition and binding to the target protein. The slide then is washed (e.g. , with reaction buffer) to remove unbound or nonspecifically bound protein specific binding moieties. In some embodiments, the protein specific binding moiety is conjugated to a detectable moiety. In other embodiments, the protein specific binding moiety is a primary antibody, which subsequently is detected by a second specific binding moiety, e.g., an anti-antibody-detectable moiety conjugate. In a working embodiment, the primary antibody was a mouse antibody, which was detected by a goat anti-mouse antibody labeled with hydroxyquinoxiline (HQ).

The detectable moiety may be detected directly or indirectly. In some embodiments, the detectable moiety is detected by adding components of a second detection system to the slide. The second detection system is not the same as the first detection system. In particular embodiments, the second detection system produces a different detectable color than the first detection system. In some embodiments, the second detection system includes an anti-label antibody-enzyme conjugate, an enzyme substrate, and optionally one or more additional components. In certain embodiments, the detectable moiety is a hapten, and the second detection system includes an anti-hapten antibody-enzyme conjugate, e.g. , a mouse anti- hapten antibody-horseradish peroxidase (HRP) conjugate. While any of the various chromogens shown in Table 1 may be used, a suitable substrate for horseradish peroxidase is diaminobenzidine, which reacts with HRP to produce a detectable brown color (precipitate).

In some embodiments, Hematoxylin II subsequently is added to the slide and incubated to produce a light-blue counterstain. The slide then is rinsed with reaction buffer and bluing reagent. The stained slide is coverslipped and viewed using brightfield microscopy.

VII. Kit for Dual Detection of miRNA and Protein Targets

A kit for dual detection of a miRNA target and a protein target that may be regulated by the miRNA are also a feature of this disclosure. Embodiments of the kit include a non-enzymatic cell conditioning solution, reagents suitable for use in an automated system for detecting a miRNA target in a sample, and reagents suitable for use in an automated system for detecting a protein target. The kit also may include instructions for performing a method to detect the miRNA and protein.

In some embodiments, reagents suitable for detecting a miRNA target include a nucleic acid specific binding moiety selected for a particular miRNA target. In one embodiment, the nucleic acid specific binding moiety is a locked nucleic acid probe-hapten conjugate. In certain embodiments, the reagents suitable for detecting the miRNA target further include an anti-hapten antibody, a secondary antibody-enzyme conjugate, and an enzyme substrate.

In some embodiments, reagents suitable for detecting a protein target include a protein specific binding moiety selected for a protein target that may be regulated by the miRNA. In one embodiment, the protein specific binding moiety is a primary antibody. In certain embodiments, the reagents suitable for detecting the protein target further include an anti-antibody-label conjugate (e.g. , an anti- antibody-hapten conjugate), an anti-label antibody-enzyme conjugate, and an enzyme substrate.

VIII. Examples

The following examples are provided to illustrate certain features of illustrative embodiments. A person of ordinary skill in the art will appreciate that the scope of the present invention is not limited to those features disclosed in these examples.

Example 1

This example provides details associated with one embodiment for automated detection of miRNA. LCS deparaffinization was conducted for three 65 °C cycles for 4 minutes. Proteinase K treatment for single stain protocol included using 10-15μg/mL solution of Proteinase K, diluted in 5mM Tris buffer pH 7.3 with ImM EDTA. Concentrations and incubation times are tissue dependent. Probe hybridization includes denaturing at 80° C, and hybridization at 30 °C less than the RNA T_m for one hour. A double DIG labeled probe was diluted in a hybridization buffer (microRNA ISH buffer lx cone, Exiqon, Woburn, MA). Two stringency washes were conducted using 2x SSC at the hybridization temperature.

The detection protocol include mouse anti-DIG at 2μg/mL for 20 minutes (Roche Applied Science p/n: 1 1333062910). When Amp-HQ was not used the conditions were anti-mouse AP for 16 minutes, and blue detection (UltraMap and

ChromoMap Blue, Ventana Medical Systems, Inc.) - 2 substrate cycles for 44 minutes. If Amp-HQ was used, then the conditions were an anti-mouse HRP (OmniMap, Ventana Medical Systems, Inc.) for 16 minutes, Amp-HQ for 24 minutes, mouse anti-HQ AP for 16 minutes, and blue detection (ChromoMap Blue, Ventana Medical Systems, Inc.) - 1 substrate cycle for 44 minutes.

Example 2

This example describes a general protocol for dual staining miR A and protein on an automated system, such as Ventana Medical Systems, Inc.'s

Discovery Ultra automated instrument. Paraffin was removed from the slides by heating the slides to 65 °C. The slides were then rinsed with a buffer solution (Discovery EZ Prep; Ventana Medical Systems, Inc. p/n 950-100) and preparatory solution (Liquid Coverslip (LCS) Ventana Medical Systems, Inc.p/n 650-010). After the paraffin was removed from the slides, the following cycle was performed. The slides were heated to 90 °C. Cell conditioning solution, a tra-based buffer with a slightly basic pH, which, at elevated temperatures, disrupts the covalent bonds formed by formalin in tissue (CC1 Ventana Medical Systems, Inc. p/n 950- 124) was applied, and the sample incubated for 8 minutes. The slides were rinsed with reaction buffer (Ventana Medical Systems, Inc. p/n 950-300) and the cell conditioning solution, CC1, was reapplied. The process was done for a total of three applications. One hundred microliters of a 7.5nM solution of a double digoxigenin-labeled LNA probe (Exiqon, Woburn, MA) was applied to the slide and was heated to 80 °C for 8 minutes before cooling to the hybridization temperature of 30 °C below the specified T_m on the Exiqon specification sheet provided with the probe. The probe hybridized on the slide for 1 hour at the hybridization temperature before being detected with 100 of a 2 μg/mL solution of a mouse anti- digoxigenin antibody solution (Roche Applied Science, Cat No. 11333062910). The blue signal used to detect the miRNA was generated using a commercially available detection kit (UltraMap anti-Mouse Blue Kit, Ventana

Medical Systems, Inc. p/n 760-156). The NBT/BCIP substrate incubated on the slide for two separate incubations of 44 minutes.

Following miRNA detection, a mouse antibody for detecting the protein of interest was applied to the slide and incubated for 32 minutes at 37 °C. After the primary antibody incubation, the slide was rinsed with reaction buffer and a goat anti-mouse secondary labeled with HQ was incubated on the slide for 16 minutes. After the secondary antibody incubation, the slide was rinsed with reaction buffer and 100 of a 25 μ§/ηιΙ. solution of a mouse anti-HQ HRP conjugate was applied to slide and incubated at 37 °C for 8 minutes.

A DAB detection kit (ChromoMap, Ventana Medical Systems, Inc.) was used to generate the signal for the protein. The slide was rinsed with reaction buffer and a hematoxylin solution (Hematoxylin II, Ventana Medical Systems, Inc. p/n 790-2208) was diluted 1 : 10 and 100 μΐ, was applied to the slide for 4 minutes. The slide was rinsed with reaction buffer and bluing reagent (Ventana Medical Systems, Inc. p/n 760-2037) was incubated on the slide for 4 minutes. The hematoxylin solution was diluted in order to clearly distinguish the blue signal generated for the miRNA from the blue counterstain. The slides were washed in soapy water to remove any LCS, dehydrated using gradient alcohols and coverslipped for viewing using brightfield microscopy.

Example 3

This example describes one embodiment of a miR-205 - HER3 dual stain protocol. Three deparaffinization cycles were conducted at 65 °C for 4 minutes.

Three cell conditioning cycles also were performed using CCl for 8 minutes. miR- 205 probe hybridization was preferentially conducted first; weaker staining occurs if miRNA probe hybridization was done after protein detection. A denaturing step was conducted at 80 °C. Thereafter, miR-205 probe hybridization was conducted at 60 °C. The double digoxigenin-labeled probe was diluted in hybridization buffer

(lx hybe buffer, Exiqon, Woburn, MA). Two stringency washes were conducted using saline-sodium citrate (SSC) at 60 °C.

miR-205 detection was then performed. A mouse anti- digoxigenin antibody was applied to the sample for 20 minutes. An anti-mouse antibody- alkaline phosphatase conjugate (UltraMap, Ventana Medical Systems, Inc.) was then applied for 16 minutes. A biotin-free NBT/BCIP detection kit (ChromoMap Blue; Ventana Medical Systems, Inc.) was then used for 2 substrate cycles for 44 minutes.

HER3 was then detected using a mouse anti-HER3 antibody with incubation at 37 °C for 32 minutes. Detection was then performed using a goat anti-mouse antibody labeled with HQ hapten, diluted to 20 μg/mL, with incubation for 16 minutes. A mouse anti-HQ-HRP conjugate, diluted to 25 μg/mL, was then applied for 8 minutes. Diaminobenzidine (DAB) was then used for visualization. A light-blue counterstain was the produced using a diluted (1 : 10) hematoxylin solution (Hematoxylin II, Ventana Medical Systems, Inc.) in deionized water. The results obtained using the protocol of this Example 3 are illustrated in FIGS. 1(A)- (C). FIG. 1(A) shows the dual stain assay described herein. FIG. 1(B) and FIG.

1(C) shows corresponding single stain procedures for HER3 and mi-R205, respectively. The cases, and replicates performed on other lobular breast carcinoma tissue samples, were highly positive for HER3. In all tissues, tumor cells with intense HER3 staining displayed mild to negative miR-205 signal.

Conversely, the myoepithelial cells were highly positive for miR-205 and showed decreased HER3 staining intensity.

Example 4

It has been reported that miR-205 is an oncosuppressor gene in breast cancer, able to interfere with the proliferative pathway mediated by HER receptor family. An increasing amount of experimental evidence shows that miRNAs can have a causal role in breast cancer tumorigenesis as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. HER2

overexpression is a hallmark of a particularly aggressive subset of breast tumors, and its activation is dependent on the trans-interaction with other members of HER family; in particular, the activation of the PBK/Akt survival pathway, so important in tumorigenesis, is predominantly driven through phosphorylation of the kinase- inactive member HER3. It is observed that miR-205 is down-modulated in breast tumors compared with normal breast tissue and understood as directly targeting the HER3 receptor with the implication that it inhibits the activation of the downstream mediator Akt.

This example describes one embodiment of a miR-205 - Bcl2 dual stain protocol. Three deparrafmization cycles were conducted at 65 °C for 4 minutes. Three cell conditioning cycles also were performed using a cell conditioning solution (CC1, Ventana Medical Systems, Inc.) for 8 minutes. miR-205 probe hybridization was preferentially conducted first; weaker staining occurs if miRNA probe hybridization was done after protein detection. A denaturing step was conducted at 80 °C. Thereafter, miR-205 probe hybridization was conducted at 60 °C. The double digoxigenin-labeled probe was diluted in hybridization buffer (lx hybe buffer, Exiqon). Two stringency washes were conducted using saline-sodium citrate (SSC) at 60 °C.

miR-205 detection was then performed by treating the sample with a mouse anti-digoxigenin antibody for 20 minutes. An anti-mouse antibody-alkaline phosphatase conjugate (Ventana Medical Systems, Inc. UltraMap) was then applied for 16 minutes. A biotin-free NBT/BCIP detection kit (ChromoMap Blue, Ventana Medical Systems, Inc.) was then used for 2 substrate cycles for 44 minutes.

Bcl2 detection was then performed using a mouse anti-bcl2 antibody with incubation at 37 °C for 32 minutes. The sample was then hybridized with a goat anti-mouse antibody labeled with an HQ hapten diluted to 20 μg/mL with incubation for 16 minutes. A mouse anti-HQ-HRP conjugate, diluted to 25 μg/mL, was then applied for 8 minutes. Diaminobenzidine (ChromoMap DAB, Ventana Medical Systems, Inc.) was then used for visualization. A light-blue counterstain was then produced using a hematoxylin solution (Hematoxylin II, Ventana Medical

Systems, Inc.) diluted to 1 : 10 in deionized water.

The results obtained using the protocol of this Example 4 are illustrated in FIGS. 2(A)-(B). In particular, these images represent two independent examples of breast specimens stained with the dual assay for miR-205 and Bcl2.

Example 5

This example describes one embodiment of a miR-126 - CR dual stain protocol. The results obtained using the protocol of this Example 4 are illustrated in FIGS. 3(A)-(D). Three deparrafmization cycles were conducted at 65 °C for 4 minutes. Three cell conditioning cycles also were performed using a cell conditioning solution (CC1, Ventana Medical Systems, Inc.) for 8 minutes. It was determined that miR-126 probe hybridization was preferentially conducted second. One rationale for this order is that when miR-126 was done first, the Goat-anti- mouse-HQ recognized the Mouse anti-DIG and showed false co-localization of the CRK with the miR-126. Another aspect of this example was the use of a mouse anti-DIG AP to avoid the use of a goat anti-mouse AP from binding to any mouse anti-HQ conjugates. CRK detection was performed using a mouse anti-CRK antibody with incubation at 37 °C for 32 minutes. The sample was then hybridized with a goat anti-mouse antibody labeled with an HQ hapten diluted to 20 μg/mL with incubation for 16 minutes. A mouse anti-HQ-HRP conjugate, diluted to 25 μg/mL, was then applied for 8 minutes. Diaminobenzidine (ChromoMap DAB, Ventana

Medical Systems, Inc.) was then used for visualization. miR-126 was then performed by first using a denaturing step conducted at 80 °C. Thereafter, miR- 126 probe hybridization was conducted at 55 °C. The double digoxigenin-labeled probe was diluted in hybridization buffer (lx hybe buffer, Exiqon). Two stringency washes were conducted using saline-sodium citrate (SSC) at 55 °C. miR-126 detection was then performed by treating the sample with a mouse anti- digoxigenin antibody for 16 minutes. An anti-mouse antibody-alkaline phosphatase conjugate (Ventana Medical Systems, Inc. UltraMap) was then applied for 16 minutes. A biotin-free NBT/BCIP detection kit (ChromoMap Blue, Ventana Medical Systems, Inc.) was then used for 1 substrate cycles for 60 minutes. A light-blue counterstain was then produced using a hematoxylin solution

(Hematoxylin II, Ventana Medical Systems, Inc.) diluted to 1 : 10 in deionized water. FIG. 3(A) shows the results of a lung section stained with the dual assay for miR-126 and CRK. FIG. 3(B) shows the results of a first control experiment performed according to this procedure that identified the CRK in the absence of the miR-126 probe. FIG. 3(C) shows the results of a second control experiment according to this procedure that identified the miR-126 in the absence of the anti- CRK antibody. Of note, the single stain of the miR-126 shown in FIG. 3(B) is darker and shows greater background than what was observed in the dual stain (FIG. 3(A)). It was observed that the detection of miR-126 after the protein detection was weaker. To counteract this weaker detection, the biotin-free

NBT/BCIP detection step was extended to 1 hour instead of 24 minutes, 24 minutes being a typical time for a single stain using the described reagent concentrations. Since the control staining procedures were uniform across the slides, the miR-126 single staining is not stained according to a preferred single staining procedure. As a control to the miR-126 probe staining, a scramble probe was used as a control. The results of this control experiment are shown in FIG. 3(D).

Example 6 - 26

Examples 6 - 26 are summarized in Table 2. Disclosed are miRNA and protein combinations within the scope of the present invention. Using methods and kits described herein, the dual detection of these combinations of miRNA and proteins is understood to provide particular insight into the specified cancers.

Table 2

Example 6: It has been shown that the highly malignant human brain tumor, glioblastoma, strongly overexpresses a specific miRNA, miR-21. Studies have shown elevated miR-21 levels in human glioblastoma tumor tissues, early- passage glioblastoma cultures, and in six established glioblastoma cell lines (A172, U87, U373, LN229, LN428, and LN308) compared with nonneoplastic fetal and adult brain tissues and compared with cultured nonneoplastic glial cells.

Knockdown of miR-21 in cultured glioblastoma cells triggers activation of caspases and leads to increased apoptotic cell death. Data has suggested that aberrantly expressed miR-21 may contribute to the malignant phenotype by blocking expression of critical apoptosis-related genes. It has also been shown through luciferase activity assays that a number of genes involved in apoptosis, PDCD4, MTAP, and SOX5, carry putative miR-21 binding sites. Inhibition of miR-21 increases endogenous levels of PDCD4 in cell line T98G and over- expression miR-21 inhibits PDCD4-dependent apoptosis. Together, these results indicate that miR-21 expression plays a key role in regulating cellular processes in glioblastomas and may serve as a target for effective therapies. Accordingly, a procedure for the dual staining of miR-21 and Caspase-3 in glioblastomas or other cancer is one embodiment of the present disclosure.

Example 7: It has been shown that miR-21 modulates biological functions of pancreatic cancer cells including their proliferation, invasion, and

chemoresistance. Due to the poor prognosis of pancreatic cancer, novel diagnostic modalities for early diagnosis and new therapeutic strategy are urgently needed. Recently, miR-21 was reported to be strongly overexpressed in pancreatic cancer as well as in other solid cancers. A miR-21 expression assessment in pancreatic cancer cell lines and pancreatic tissue samples performed using quantitative real-time reverse transcription-PCR amplification has been reported. It is known that miR- 21 is markedly overexpressed in pancreatic cancer cells compared with

nonmalignant cells, and miR-21 in cancer tissues was much higher than in nonmalignant tissues. Cancer cells trans fected with a miR-21 precursor showed significantly increased proliferation, Matrigel invasion, and chemoresistance for gemcitabine compared with control cells. In contrast, inhibition of miR-21 decreased proliferation, Matrigel invasion, and chemoresistance for gemcitabine. Moreover, miR-21 positively correlated with the mRNA expression of invasion- related genes, matrix metalloproteinase-2 and -9, and vascular endothelial growth factor. This data suggest that miR-21 expression is increased in pancreatic cancer cells and that miR-21 contributes to the cell proliferation, invasion, and

chemoresistance of pancreatic cancer.

Further studies have shown that the antisense inhibition of microRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma. These studies have demonstrated the contribution of overexpressed miR-21 and -221 to the malignant phenotype using antisense oligonucleotides. The results indicate that low nanomolar concentrations of both antisense oligonucleotides reduced proliferation of pancreatic cancer cell lines. It is now known that protein levels of tumor suppressor targets of the miRNAs were increased by antisense to miR-21 (PTEN and RECK) and miR-221 (p27).

Antisense to miR-21 and miR-221 sensitized the effects of gemcitabine, and the antisense-gemcitabine combinations were synergistic at high fraction affected.

It is also understood that miR-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). Studies have shown that miR-21 is overexpressed in tumor tissues compared with the matched normal tissues. Reports using two- dimensional differentiation in-gel electrophoresis of tumors treated with anti-miR- 21 identified the tumor suppressor tropomyosin 1 (TPM1) as a potential miR-21 target. There appears to be a putative miR-21 binding site at the 3 '-untranslated region (3'-UTR) of TPM1 variants VI and V5. Reports of a western blot with the cloned TPM1-V1 plus the 3'-UTR suggest that TPM1 protein level was also regulated by miR-21, whereas real-time quantitative reverse transcription-PCR was found without difference at the mRNA level, suggesting translational regulation. The notion that miR-21 functions as an oncogene is supported by the down- regulation of TPM1 by miR-21. Yet other reports have linked miR-21 to programmed cell death 4 (PDCD4) and the p53 tumor suppressor protein.

Specifically, miR-21 has been shown to post-transcriptionally down-regulate tumor suppressor PDCD4 and stimulate invasion, intravasation and metastasis in colorectal cancer. Tumor-suppressor PDCD4 inhibits transformation and invasion and is downregulated in cancers. Accordingly, a procedure for the dual staining of miR-21 and one or more proteins selected from the group consisting of FINRK, p53-related Tap63, PDCD4, PTEN, RECK, and TPM1 in glioblastoma, pancreatic cancer, breast cancer, colorectal cancer, or other cancer is one embodiment of the present disclosure.

Example 8: It has been reported that the PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo. It is known that activated oncogenic signaling is central to the development of nearly all forms of cancer, including glioma. Research has revealed the importance of the Akt pathway and its molecular antagonist PTEN in the process of gliomagenesis. The identification of miR-26a as a direct regulator of PTEN expression has been reported. It has also been shown that miR-26a is frequently amplified at the DNA level in human glioma, most often in association with monoallelic PTEN loss. It has also been shown that miR-26a-mediated PTEN repression in a murine glioma model both enhances de novo tumor formation and precludes loss of heterozygosity and the PTEN locus. Accordingly, a conclusion regarding an epigenetic mechanism for PTEN regulation in glioma has been recognized. Accordingly, a procedure for the dual staining of miR-26a and PTEN in glioblastoma or other cancer is one embodiment of the present disclosure.

Example 9/10: It has been reported that miRNA 221/222 regulates p27^Kipl in glioblastoma. Levels of p27^Kipl, a key negative regulator of the cell cycle, are often decreased in cancer. In most cancers, levels of p27^Kipl mRNA are unchanged and increased proteolysis of the p27^Kipl protein is thought to be the primary mechanism for its downregulation. It has been shown that p27^Kipl protein levels are also downregulated by miRNA 221/222 in cancer cells, which have been shown to be upregulated in glioblastoma relative to adjacent normal brain tissue. The genes for miRNA 221 and miRNA 222 occupy adjacent sites on the X

chromosome. It has been suggested that their expression is coregulated and that they have the same target specificity. Antagonism of either miRNA 221 or 222 in glioblastoma cells also caused an increase in p27^Kipl levels and enhanced expression of the luciferase reporter gene fused to the p27^Kipl 3'UTR. The data has shown that p27^Kipl is a direct target for miRNAs 221 and 222, and suggest a role for these microRNAs in promoting the aggressive growth of human glioblastoma. It has been also reported that antisense inhibition of miRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma.

These reports linked the contribution of overexpressed miR-21 and -221 to the malignant phenotype by inhibiting the miRNAs using antisense oligonucleotides.

In particular, low nanomolar concentrations of both antisense oligonucleotides reduced proliferation of pancreatic cancer cell lines. Reduced proliferation was less pronounced in the normal ductal epithelial cell line human pancreatic Nestin- expressing cell or in pancreatic cancer cell lines exposed to an irrelevant control oligonucleotide. Inhibition of miR-21 and miR-221 increased the amount of apoptosis in HS766T cells by 3- to 6-fold compared with the control

oligonucleotide. HS766T cells exposed to miR-21 antisense resulted in cell cycle arrest (Gl phase). Protein levels of tumor suppressor targets of the miRNAs were increased by antisense to miR-21 (PTEN and RECK) and miR-221 (p27).

Antisense to miR-21 and miR-221 sensitized the effects of gemcitabine. These reports indicate that antisense to miR-21 and miR-221 results in significant cell killing under various conditions and that antisense oligonucleotides targeted to miRNA represents a potential new therapy for pancreatic cancer. Accordingly, a procedure for the dual staining of miR-221 and/or miR-222 and p27^Kipl, PTEN, and/or RECK in glioblastoma, pancreatic cancer, or other cancer is one

embodiment of the present disclosure.

Example 11: It has been reported that miR-7 is down-regulated in glioblastoma and that miR-7 inhibits the epidermal growth factor receptor and the Akt pathway. miR-7 is a potential tumor suppressor in glioblastoma because it is involved in various cancer pathways. According to one understanding, miR-7 suppresses epidermal growth factor receptor expression and independently inhibits the Akt pathway via targeting upstream regulators. miR-7 expression was down- regulated in glioblastoma versus surrounding brain, with a mechanism involving impaired processing. Transfection with miR-7 decreased viability and invasiveness of primary glioblastoma lines. Accordingly, it is our understanding that miR-7 is a regulator of major cancer pathways and suggests that it has therapeutic potential for glioblastoma. Accordingly, a procedure for the dual staining of miR-7 and Akt or a related Akt pathway protein in glioblastoma or other cancer is one embodiment of the present disclosure.

Example 12: Other reports have identified miR-34a as inhibiting glioblastoma growth by targeting multiple oncogenes. miR-34a is a transcriptional target of p53 that is down-regulated in some cancer cell lines. Transfection of miR-34a is known to down-regulate c-Met in human glioma and meduUoblastoma cells and Notch- 1, Notch-2, and CDK6 protein expressions in glioma cells.

Furthermore, miR-34a expression is understood to inhibit c-Met reporter activities in glioma and meduUoblastoma cells and Notch- 1 and Notch-2 3 '-untranslated region reporter activities in glioma cells and stem cells. Analyses of human specimens have shown that miR-34a expression is down-regulated in glioblastoma tissues as compared with normal brain and in mutant p53 gliomas as compared with wild-type p53 gliomas. Furthermore, miR-34a levels in human gliomas have been inversely correlated to c-Met levels measured in the same tumors. Transient transfection of miR-34a into glioma and meduUoblastoma cell lines were shown to strongly inhibit cell proliferation, cell cycle progression, cell survival, and cell invasion. Transfection of miR-34a into human astrocytes was not shown to affect cell survival and cell cycle status. Forced expression of c-Met or Notch- 1 /Notch-2 transcripts lacking the 3 '-untranslated region sequences were shown to partially reverse the effects of miR-34a on cell cycle arrest and cell death in glioma cells and stem cells, respectively. As such, it has been reported that miR-34a suppresses brain tumor growth by targeting c-Met and Notch. Transcription of the three miRNA miR-34 family members was also found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells. miR-34a has also been reported to inhibit migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells. Further, reports have shown that miR-34a regulates silent information regulator 1 (SIRT1) expression. miR-34 inhibition of SIRT1 leads to an increase in acetylated p53 and expression of p21 and PUMA, transcriptional targets of p53 that regulate the cell cycle and apoptosis, respectively. Furthermore, miR-34 suppression of SIRT1 ultimately leads to apoptosis in WT human colon cancer cells but not in human colon cancer cells lacking p53. miR-34a is also understood to be a transcriptional target of p53, suggesting a positive feedback loop between p53 and miR-34a. Thus, miR-34a functions as a tumor suppressor, in part, through a SIRTl-p53 pathway. Accordingly, a procedure for the dual staining of miR-34a and Notch- 1, c-MET, Bcl-2, Notch-1, CdK4, Sirtl in glioblastoma, hepatocellular carcinoma, pancreatic cancer, colon cancer, or other cancer is one embodiment of the present disclosure.

Example 13: The microRNA miR-326 is understood to act in a feedback loop with Notch and may have therapeutic potential against brain tumors. The Notch pathway plays key roles in nervous system development and in brain tumors. The Notch pathway is implicated in gliomas. miR-326 was upregulated following Notch-1 knockdown; thus, it is not only suppressed by Notch but also inhibits Notch proteins and activity, indicating a feedback loop. miR-326 has been observed as downregulated in gliomas via decreased expression of its host gene. Transfection of miR-326 into both established and stem cell-like glioma lines was cytotoxic, and rescue was obtained with Notch restoration. Accordingly, a procedure for the dual staining of miR-326 and Notch-1 in glioblastoma or other cancer is one embodiment of the present disclosure.

Example 14: It is understood that miR-128 inhibits glioma proliferation and self-renewal. miR expression profiling of human glioblastoma specimens versus adjacent brain devoid of tumor has revealed several significant alterations, including a pronounced reduction of miR-128 in tumor samples. It has been observed that miR-128 expression significantly reduced glioma cell proliferation in vitro and glioma xenograft growth in vivo; miR-128 caused a striking decrease in expression of the Bmi-1 oncogene. In a panel of patient glioblastoma specimens,

Bmi-1 expression was significantly up-regulated and miR-128 was down-regulated compared with normal brain. It is understood that Bmi-1 functions in epigenetic silencing of certain genes through epigenetic chromatin modification; miR-128 expression caused a decrease in histone methylation and Akt phosphorylation, and up-regulation of p21(CIPl) levels, consistent with Bmi-1 down-regulation. As Bmi-1 has been shown to promote stem cell self-renewal and miR-128 has been shown to specifically block glioma self-renewal consistent with Bmi-1 down- regulation. Accordingly, a procedure for the dual staining of miR-128 and Bmi-1 in glioblastoma or other cancer is one embodiment of the present disclosure.

Example 15/16: It has been reported that miR-141 and miR-200c are significantly down-regulated in renal cell carcinomas (clear cell carcinomas, "CCC," and chromophobe renal cell carcinomas , "ChCC"). It has been shown that

43 miRNAs are differentially expressed between CCC and normal kidney, of which 37 were significantly down-regulated in CCC and the other 6 were up- regulated. Fifty-seven (57) miRNAs were differentially expressed between ChCC and normal kidney, of which 51 were significantly down-regulated in ChCC and the other 6 were up-regulated. These observations indicate that expression of miRNAs tends to be down-regulated in both CCC and ChCC compared with normal kidney. ZFHXIB, a transcriptional repressor for CDHl/E-cadherin, has been shown to be up-regulated. Overexpression of miR-141 and miR-200c appears to cause down-regulation of ZFHXIB and up-regulation of E-cadherin in two renal carcinoma cell lines, ACHN and 786-0. As such, the understanding is that down- regulation of miR-141 and miR-200c in CCCs might be involved in suppression of CDH1 /E-cadherin transcription via up-regulation of ZFHXIB.

Further reports indicate that a reciprocal repression between ZEBl and members of the miR-200 family promotes EMT and invasion in cancer cells. The embryonic programme 'epithelial-mesenchymal transition' (EMT) is thought to promote malignant tumor progression. The transcriptional repressor zinc-finger E- box binding homeobox 1 (ZEBl) is an inducer of EMT in various human tumors, and has been shown to promote invasion and metastasis of tumor cells. ZEBl reportedly directly suppresses transcription of micro RNA-200 family members miR-141 and miR-200c, which strongly activate epithelial differentiation in pancreatic, colorectal and breast cancer cells. The EMT activators transforming growth factor beta2 and ZEBl appear to be the predominant targets down-regulated by these microRNAs. These reports indicate that ZEBl may trigger a microRNA- mediated feed-forward loop that stabilizes EMT and promotes invasion of cancer cells. According to another explanation, this loop might switch and induce epithelial differentiation. Accordingly, a procedure for the dual staining of miR- 200c and/or miR-141 and Bmi-1, ZEBl, and/or ZEB2 in renal cell carcinoma, pancreatic cancer, breast cancer or other cancer is one embodiment of the present disclosure.

Example 17: It has been reported that RAS is regulated by the let-7 miRNA family. In particular, it is understood that the let-7 family negatively regulates let-60/RAS, loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. miR-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. It has been reported that let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, indicating a possible mechanism for let-7 in cancer. Accordingly, a procedure for the dual staining of let-7 or a family member and RAS in lung cancer or other cancer is one embodiment of the present disclosure.

Example 18: It has also been reported that Let-7 expression defines two differentiation stages of cancer. Within an ovarian cancer model, it has been shown that expression of let-7 and high-mobility group A2 (HMGA2) is a better predictor of prognosis than classical markers such as E-cadherin, vimentin, and Snail. Loss of let-7 expression is a marker for less differentiated cancer.

Accordingly, a procedure for the dual staining of let-7 or a family member and HMGA2 in lung cancer, ovarian cancer, or other cancer is one embodiment of the present disclosure.

Example 19: miR-15a and miR-16 are understood to be implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down- regulated in non- small cell lung cancer. miR-15a/miR-16 are frequently deleted or down-regulated in squamous cell carcinomas and adenocarcinomas of the lung. In these tumors, expression of miR-15a/miR-16 inversely correlates with the expression of cyclin Dl . In non-small cell lung cancer (NSCLC) cell lines, cyclins

Dl, D2, and El are directly regulated by physiologic concentrations of miR- 15a/miR-16. Consistent with these results, overexpression of these miRNAs induces cell cycle arrest in G(1)-G(0). The reports indicate that miR-15a/miR-16 are implicated in cell cycle control and likely contribute to the tumorigenesis of NSCLC.

Furthermore, miR-15a-miR-16-l cluster has been reported as controlling prostate cancer by targeting multiple oncogenic activities. In particular, miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13ql4, which is frequently deleted in cancer. The miR-15a and miR-16-1 cluster have been reported to target CCND1 (encoding cyclin Dl) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely up- regulated. miR-15a and miR-16 may act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion.

Accordingly, a procedure for the dual staining of miR-15 and/or miR-16 and a protein selected from the group consisting of Cyclin Dl, Cyclin D2, Cyclin El, Bcl-2, WNT3A and combinations thereof in lung cancer, prostate cancer, or other cancer is one embodiment of the present disclosure.

Example 20: It is understood that the up-regulation of several miR-1 targets including FoxPl, MET, and HDAC4 in primary human hepatocellular carcinoma (HCC) and down-regulation of their expression in 5 -AzaC -treated HCC cells implicates miR-1 and its targets as having a role hepatocarcinogenesis.

Accordingly, a procedure for the dual staining of miR-1 and a protein selected from the group consisting of c-MET, Pim-1, FoxPl, HDAC4 and combinations thereof in lung cancer, hepatocellular carcinoma, or other cancer is one embodiment of the present disclosure.

Example 21: It has been reported that a loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. It is known that miR-122 is specifically repressed in a subset of primary tumors that are characterized by poor prognosis. Furthermore, the loss of miR-122 expression was shown in tumor cells segregates with specific gene expression profiles linked to cancer progression, namely the suppression of hepatic phenotype and the acquisition of invasive properties. Evidence suggests that miR-122 is under the transcriptional control of HNF1A, HNF3A and FTNF3B and that loss of miR-122 expression results in an increase of cell migration and invasion and that restoration of miR-122 reverses this phenotype. It was similarly reported that miR- 122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. Further, a distintegrin and metalloprotease family 10 p (ADAM 10), serum response factor (SRF), and insulin- like growth factor 1 receptor (IgflR) were validated as targets of miR-122 and were repressed by the miRNA. Yet other studies indicate that cyclin Gl is a target of miR- 122a.

Accordingly, a procedure for the dual staining of miR-122 and a protein selected from the group consisting of c-MET, IGF1R, cyclin G and combinations thereof in hepatocellular carcinoma or other cancer is one embodiment of the present disclosure.

Example 22: Reports have indicated that miR- 143, -145, and the target gene of ER 5 are associated with oncogenesis in colon and other cancer cells. In particular, it is understood that that that miR- 143 and -145 expression levels were extremely reduced in colon cancer cells and commonly in the other kinds of cancer cells tested. Similarly, miR- 145 has been associated with the down-regulation of insulin receptor substrate- 1 (IRS-1) and type 1 insulin- like growth factor receptor (IGF-IR), suggesting miR- 145 is a tumor suppressor. Accordingly, a procedure for the dual staining of miR- 143 and/or miR- 145 and a protein selected from the group consisting of IRS-1, IGF-IR, and combinations thereof in colon cancer or other cancer is one embodiment of the present disclosure.

Example 23: It has been reported that, using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR- 125 a or miR- 125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level.

Additionally, phosphorylation of ER l/2 and AKT was suppressed in SKBR3 cells overexpressing either miR- 125 a or miR- 125b. Accordingly, a procedure for the dual staining of miR- 125 and a protein selected from the group consisting of ErbB2 (Her2), ErbB3 (Her3), BMPR1, HuR and combinations thereof in breast cancer or other cancer is one embodiment of the present disclosure. Example 24: It is understood that miR-31 uses multiple mechanisms to oppose metastasis, in particular breast cancer metastasis. The expression of miR- 31 was found to correlate inversely with metastasis in human breast cancer patients. Overexpression of miR-31 in otherwise-aggressive breast tumor cells suppresses metastasis. One suggestion is that miR-31 represses metastasis-promoting genes, including RhoA. Accordingly, a procedure for the dual staining of miR-31 and a protein selected from the group consisting of FZD3, ITGA5, MMP16, RDX, RhoA and combinations thereof in breast cancer or other cancer is one embodiment of the present disclosure.

Example 25: It has been reported that the genomic loss of miR-101 leads to the overexpression of histone methyltransferase EZH2 (Enhancer of zeste homo log 2) in cancer, particularly prostate cancer. The overexpression of EZH2 and concomitant dysregulation of epigenetic pathways is implicated in cancer progression. EZH2 is overexpressed in aggressive solid tumors by mechanisms that remain unclear. However, it is now known that the expression and function of

EZH2 in cancer cell lines are inhibited by miR-101. Accordingly, a procedure for the dual staining of miR-101 and EZH2 in prostate cancer or other cancer is one embodiment of the present disclosure.

Example 26: It is understood that miR-133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer. The expression of miR-133B was reported as being 28-fold lower in lung tumor tissue compared to adjacent uninvolved tissue. It is understood that miR-133B directly targets the 3'UTRs of both MCL-1 and BCL2L2 and that apoptosis is induced following gemcitabine exposure in these tumor cells with an over-expression of miR-133B. Accordingly, a procedure for the dual staining of miR-133 and MCL-1, BCL2L2, or both in lung cancer or other cancer is one embodiment of the present disclosure.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

PATENT CLAIMS

1. An automated method for dual detection of a miRNA target and a protein target within a tissue sample, the method comprising:

applying to the tissue sample reagents suitable for detecting the miRNA target in the sample using an automated staining apparatus;

applying to the tissue sample reagents suitable for detecting the protein target using the automated staining apparatus; and

detecting the miRNA target and the protein target.

2. The method of claim 1 , wherein the miRNA target and the protein target are biologically related.

3. The method of claim 1, wherein detecting the miRNA target and the protein target includes microscopically examining the tissue for dual staining within a contextual framework of the sample.

4. The method of claim 1 , wherein the method is devoid of a protease cell conditioning step.

5. The method of claim 1, further comprising applying to the tissue sample a non-enzymatic cell conditioning reagent at a temperature ranging from about 80 °C to about 95 °C.

6. The method of claim 5, wherein the non-enzymatic cell conditioning reagent is a buffer having a basic pH.

7. The method of claim 5, wherein the non-enzymatic cell conditioning reagent comprises a Tris-based buffer having a pH of from about 7.7 to about 9 at a temperature greater than ambient.

8. The method of claim 1, further comprising:

applying to the tissue sample a non-enzymatic cell conditioning reagent; contacting the sample with a nucleic acid specific binding moiety selected for a particular miRNA target;

detecting the nucleic acid specific binding moiety;

contacting protein in the sample with a protein specific binding moiety selected for a protein target; and

detecting the protein specific binding moiety.

9. The method of claim 1 , wherein the miRNA target is selected from let-7, let-7a, let 7a- 1, let-7b, let-7b-l, let-7c, let-7d, let-7g, miR-1, miR-l-d, miR-1 - 2, miR-7, (hsa-miR-7- 1 - hsa-miR-7-3), (has-miR-9- 1 - hsa-miR-9-3), miR-9, miR-lOa, miR-lOb, miR-15, miR-15a, miR-15b, miR-16, miR-16-1, miR-16-2, miR-17, miR-17-3p, miR-18a, miR-18b, miR-19a, miR19b-l, miR19b-2, miR-20a, miR-20b, miR-21, miR-22, miR-23, miR-23a, miR-23b, miR-24, miR-25, miR- 26a, miR-27a, miR-27b, miR-28, miR-29a, miR-29b, miR-29c, miR-30a-3p, miR- 30a, miR-30b, miR-30c, miR-30e-5p, miR-31, miR-32, miR-33, miR-34a, miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a, miR-100, miR-101, miR-103, miR- 105, miR-106b, miR-107, miR-108, miR-122, miR-124, miR-125, miR-125b, miR- 126, miR-127, miR-128, miR-129, miR-130, miR-130a, miR-132, miR-133, miR- 133a, miR-133a-2, miR-133b, miR-134, miR-135, miR-136, miR-137, miR-138, miR-139, miR-140, miR-141, miR-142, miR-143, miR-144, miR-145, miR-146a, miR-147, miR-148a, miR-149, miR-150, miR-151, miR-152, miR-153, miR-154, miR-155, miR-181a, miR-182, miR-183, miR-184, miR-185, miR-186, miR-187, miR-188, miR-190, miR-191, miR-192, miR-193, miR-194, miR-195, miR-196a, miR-197, miR-198, miR-199, miR-199a-l, miR-200b, miR-200c, miR-201, miR- 203, miR-204, miR-205, miR-206, miR-207, miR-208, miR-210, miR-211, miR- 212, miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-219, miR- 221, miR-222, miR-223, miR-224, miR-291-3p, miR-292, miR-292-3p, miR-293, miR-294, miR-295, miR-296, miR-297, miR-298, miR-299, miR-300, miR-301, miR-320, miR-321, miR-322, miR-323, miR-324, miR-325, miR-326, miR-328, miR-329, miR-330, miR-331, miR-333, miR-335, miR-337, miR-338, miR-339, miR-340, miR-341, miR-342, miR-344, miR-345, miR-346, miR-350, miR-361, miR-362, miR-363, miR-365, miR-367, miR-368, miR-369, miR-370, miR-371, miR-373, miR-380-3p, miR-409, miR-410, or miR-412, or functional variants thereof.

10. The method of claim 1, wherein the protein target is selected from HER family, Ras, Myc, ANP32A, and SMARCA4.

11. The method of claim 1 , wherein:

the miRNA target is miR-205, and the protein target is HER2, HER3, or combinations thereof;

the miRNA target is miR-9 and/or Let-7a and the protein target is Myc; the miRNA target is Let-7 and/or miR-18a and the protein target is K-Ras and/or HMGA2;

the miRNA target is miR-196a and the protein target is Annexin Al;

the miRNA target is miR-15a and the protein target is CNOY7;

the miRNA target is miR-15b and the protein target is LASS2;

the miRNA target is miR-143 and the protein target is ING4;

the miRNA target is miR-155 and the protein target is Gabl;

the miRNA target is miR-145 and the protein target is COL3A1;

the miRNA target is miR-21 and the protein target is Caspase-3, HNRK, p53-related Tap63, PDCD4, PTEN, RECK, or TPM1;

the miRNA target is miR-221and/or miR-222 and the protein target is _p27kipl .

the miRNA target is miR-7 and the protein target is EGFR;

the miRNA target is miR-34a and the protein target is Notch- 1, c-MET, Bcl-2, Notch-l; CdK4, or Sirtl;

the miRNA target is miR-326 and the protein target is Notch- 1;

the miRNA target is miR-128 and/or miR-200c and the protein target is

Bmi-1;

the miRNA target is miR-200c and/or miR-141 and the protein target is ZEB2 and/or ZEB1;

the miRNA target is miR-15 or miR-16 and the protein target is Cyclin Dl, Cyclin D2, Cyclin El, Bcl-2, and/or WNBA; the miRNA target is miR-326 and the protein target is Notch- 1;

the miRNA target is miR-122 and the protein target is c-MET, IGF1R, and/or cyclin Gl;

the miRNA target is miR-145 and/or miR-143 and the protein target is Erk5, IGF1R, EGFR, and/or IRS-1;

the miRNA target is miR-125 and the protein target is Her2, Her3, BMPRl, and/or HuR;

the miRNA target is miR-31 and the protein target is FZD3, ITGA5,

MMP16, RDX, and/or RhoA;

the miRNA target is miR-101 and the protein target is Ezh2; or

the miRNA target is miR-133B and the protein target is MCL-1 and/or

BCL2L2;

12. The method of claim 1, wherein the tissue sample is disposed on a single slide and detecting the miRNA and protein targets occurs on the tissue sample disposed on the single slide.

13. The method of claim 1, wherein detecting the miRNA target occurs prior to detecting the protein target.

14. The method of claim 1 , wherein the dual detection is dual chromogenic detection.

15. The method of claim 1, wherein the reagents suitable for detecting the miRNA target and the reagents suitable for detecting the protein target use a label, the label being an enzyme, a hapten, or combination thereof.

16. The method of claim 15, wherein the hapten is selected from an oxazole, a pyrazole, a thiazole, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azoaryl, a benzodiazepine, or combinations thereof.

17. The method of claim 15, wherein the hapten is selected from digoxigenin, dinitrophenyl, biotin, fluorescein, rhodamine, bromodeoxyuridine, mouse immunoglobulin, or combinations thereof.

18. The method of claim 15, wherein the hapten is detected by an anti- hapten antibody.

19. The method of claim 18, wherein the anti-hapten antibody is detected by an anti-species antibody-enzyme conjugate.

20. The method of claim 19, wherein the enzyme of the anti-species antibody-enzyme conjugate is alkaline phosphatase or horseradish peroxidase.

21. The method of claim 8, wherein contacting the sample with a nucleic acid specific binding moiety selected for a particular miRNA target comprises contacting the sample with a locked nucleic acid probe.

22. The method of claim 21 , wherein the nucleic acid specific binding moiety is a conjugate comprising a hapten conjugated to the locked nucleic acid probe.

23. The method of claim 22, wherein contacting the sample with a nucleic acid specific binding moiety selected for the miRNA target comprises contacting the sample with a digoxigenin-labeled locked nucleic acid probe selected for the miRNA target.

24. The method of claim 23, further comprising:

contacting the sample with a mouse anti-digoxigenin antibody;

contacting the sample with a conjugate comprising an anti-mouse antibody conjugated to alkaline phosphatase; and

contacting the sample with an alkaline phosphatase substrate system.

25. The method of claim 8, wherein the protein specific binding moiety is a primary antibody.

26. The method of claim 25, wherein the primary antibody is detectable by a second specific binding moiety.

27. The method of claim 26, wherein the second specific binding moiety is a secondary anti-antibody.

28. The method of claim 27, wherein the secondary anti-antibody is conjugated to a hapten.

29. The method of claim 28, further comprising contacting the sample with an anti-hapten antibody-enzyme conjugate.

30. The method of claim 29, comprising contacting the sample with an enzyme substrate.

31. The method of claim 8, wherein the protein specific binding moiety selected for a protein target is a primary antibody selected for a protein target of interest, and wherein detecting the protein specific binding moiety comprises: contacting the sample with a secondary anti-antibody-hapten conjugate; contacting the sample with an anti-hapten antibody-enzyme conjugate; and contacting the sample with an enzyme substrate system.

32. The method of claim 31 , wherein the enzyme is alkaline

phosphatase or horseradish peroxidase.

33. The method of claim 32, wherein the enzyme is alkaline

phosphatase and the substrate is nitro blue tetrazolium chloride/(5-bromo-4-chloro- lH-indol-3-yl)dihydrogen phosphate (NBT/BCIP).

34. The method of claim 32, wherein the enzyme is horseradish peroxidase and the substrate is diaminobenzidine.

35. The method of claim 8, wherein the miRNA target is detected using alkaline phosphatase, NBT/BCIP, and the protein target is detected using diaminobenzidine.

36. The method of claim 1, wherein the reagents suitable for detecting the miRNA target or the reagents suitable for detecting the protein target comprise tyramide conjugates.

37. The method of claim 1, comprising:

deparaffinizing the tissue sample;

performing non-enzymatic cell conditioning;

contacting the sample with a hapten-labeled LNA probe selected for the miRNA target;

heating the sample and then cooling to a hybridization temperature below the T_m for the LNA probe;

hybridizing the sample with the probe at the hybridization temperature; contacting the sample with an anti-hapten antibody;

contacting the sample with an anti-antibody-enzyme conjugate;

treating the sample with an enzyme substrate suitable for visualizing the miRNA target;

contacting the sample with a primary antibody selected for detecting the protein target;

contacting the sample with a secondary anti-antibody labeled with at least one hapten;

contacting the sample with anti-hapten antibody-enzyme conjugate; and contacting the sample with an enzyme substrate suitable for visualizing the protein target.

38. The method of claim 37, wherein the method is devoid of submerging the sample in a bath.

39. The method of claim 37, wherein the method is devoid of applying a protease to the sample.

40. The method of claim 37, further comprising:

applying a hematoxylin stain after contacting the sample with an enzyme substrate suitable for visualizing the protein target; and

applying a bluing reagent.

41. An automated method for dual detection of miRNA and a protein that may be regulated by the miRNA in a tissue sample, comprising:

performing non-enzymatic cell conditioning on the sample;

contacting the sample with a nucleic acid probe-hapten conjugate suitable for detecting an miRNA target;

contacting the sample with an anti-hapten antibody-enzyme conjugate; contacting the sample with an enzyme substrate to visualize the miRNA; contacting the sample with a primary antibody suitable for detecting a protein target in the sample;

contacting the sample with a secondary anti-antibody enzyme conjugate; and

contacting the sample with an enzyme substrate to visualize the protein target.

42. The method of claim 41 , wherein cell conditioning consists essentially of treating the sample with a basic buffer cell conditioning fluid at a temperature ranging from about 80 °C to about 100 °C.

43. The method of claim 41 , wherein the miRNA targets are selected from Let 7a, let 7a- 1, let 7b, let 7b- 1, let-7c, let-7d, let 7g, miR-1, miR-l-d, miR-1- 2, (hsa-miR-7-1 - hsa-miR-7-3), (has-miR-9-1 - hsa-miR-9-3), miR-lOa, miR-10b, miR- 15a, miR-15b, miR-16-1, miR-16-2, miR-17, miR-17-3p, miR-18a, miR- 18b, miR- 19a, miR19b-l, miR19b-2, miR-20a, miR-20b, miR-21, miR-22, miR-23, miR-23a, miR-23b, miR-24, miR-25, miR-26a, miR-27a, miR-27b, miR-28, miR- 29a, miR-29b, miR-29c, miR-30a-3p, miR-30a, miR-30b, miR-30c, miR-30e-5p, miR-31 , miR-32, miR-33, miR-34a, miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a, miR-100, miR-101, miR-103, miR-105, miR-106b, miR-107, miR-108, miR-122, miR-124, miR-125, miR-125b, miR-126, miR-127, miR-128, miR-129, miR-130, miR-130a, miR-132, miR-133, miR-133a, miR-133a-2, miR-133b, miR- 134, miR-135, miR-136, miR-137, miR-138, miR-139, miR-140, miR-141, miR- 142, miR-143, miR-144, miR-145, miR- 146a, miR-147, miR- 148a, miR-149, miR-

150, miR-151, miR-152, miR-153, miR-154, miR-155, miR-181a, miR-182, miR- 183, miR-184, miR-185, miR-186, miR-187, miR-188, miR-190, miR-191, miR- 192, miR-193, miR-194, miR-195, miR-196a, miR-197, miR-198, miR-199, miR- 199a-l, miR-200b, miR-201, miR-203, miR-204, miR-205, miR-206, miR-207, miR-208, miR-210, miR-211, miR-212, miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-219, miR-221, miR-222, miR-223, miR-224, miR-291-3p, miR-292, miR-292-3p, miR-293, miR-294, miR-295, miR-296, miR-297, miR-298, miR-299, miR-300, miR-301, miR-320, miR-321, miR-322, miR-323, miR-324, miR-325, miR-326, miR-328, miR-329, miR-330, miR-331, miR-333, miR-335, miR-337, miR-338, miR-339, miR-340, miR-341, miR-342, miR-344, miR-345, miR-346, miR-350, miR-361, miR-362, miR-363, miR-365, miR-367, miR-368, miR-369, miR-370, miR-371, miR-373, miR-380-3p, miR-409, miR-410, or miR- 412, miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and miR- 99b, oan-miR-205, cin-miR-126, and dre-miR-126 or functional variants thereof.

44. A kit for automated dual detection of miRNA and a protein that may be regulated by the miRNA, comprising:

a non-enzymatic cell conditioning solution;

reagents suitable for use in an automated system for detecting an miRNA target in a sample; and

reagents suitable for use in an automated system for detecting a protein target in a sample.

45. The kit according to claim 44 wherein the reagents suitable for use in an automated system for detecting an miRNA target in a sample comprise a locked nucleic acid-hapten conjugate.

46. The kit according to claim 45 further comprising an anti-hapten antibody-enzyme conjugate, and a substrate for the enzyme.

47. The kit according to claim 44 wherein the reagents suitable for use in an automated system for detecting a protein target in a sample comprise a primary antibody, a secondary anti-antibody-enzyme conjugate, and an enzyme substrate.

48. A computer readable media comprising instructions for performing the method of claim 1.

49. A computer readable media comprising instructions for performing the method of claim 37.

50. A computer readable media comprising instructions for performing the method of claim 41.

51. An automated method of treating a sample mounted on a substrate to visualize expression of a miRNA and a protein within the context of the sample, the method comprising the steps of:

placing the substrate upon which the sample is mounted on an automated instrument,

applying a preparatory reagent so that the preparatory reagent contacts the sample and the miRNA and the protein within the sample are expressed and preserved,

applying detection reagents for the miRNA and the protein, and applying at least two chromogenic reagents so that the expression of the miRNA and the protein are visualized;

wherein the expression of the miRNA and the protein within the sample are evident within the context of the sample.

52. The automated method of claim 51 wherein the method is devoid of an enzymatic preparatory reagent.

53. The automated method of claim 51 wherein the step of applying the preparatory reagent enables visualization of the miRNA and the protein expression while maintaining sample morphology.

54. The automated method of claim 51 , wherein the step of applying the preparatory reagent consists essentially of applying a non-enzymatic cell conditioning reagent at a temperature ranging from about 80 °C to about 95 °C for a predetermined time.

55. The automated method of claim 51 , wherein the preparatory reagent is a non-enzymatic buffer having a slightly basic pH.