Automated RNA detection using labeled 2'-O-methyl RNA oligonucleotide probes and a signal amplification system

Abstract

A system is provided for detecting differential expression of a given miRNA in cancer cells or surrounding normal tissue in the tumor microenvironment. [Solution] A system for bright-field in situ detection of a cellular RNA target is provided, comprising: a target probe comprising a unique 2'-O-methyl RNA probe specific to the target RNA, wherein the 2'-O-methyl RNA probe is conjugated to at least one detectable moiety; and a reactive chromogen conjugate system effective for signal amplification, wherein the reactive chromogen conjugate system is adapted to bind to the detectable moiety of the target probe.

Description

[Technical field]

[0001] This disclosure relates to an automated method for sensitive and specific detection of any cellular RNA molecule, including microRNA, messenger RNA and non-coding RNA. The technology includes probe design for the detection of RNA molecules in formalin-fixed paraffin-embedded tissue (FFPET) samples and the use of the probes in an automated format. [Background technology]

[0002] The human genome encodes over a thousand short (17-24 bases) non-coding microRNA molecules that regulate diverse cellular functions. Some of these functions include cell proliferation, motility, and immune surveillance, and are known to be involved in tumorigenesis. Not surprisingly, numerous clinical studies suggest that many microRNAs will be strong prognostic and / or predictive biomarkers for a variety of diseases, including breast, colorectal, lung, and prostate cancer. However, due to their short sequence length, technical obstacles exist for the detection of these potential biomarker targets: (i) limited target sequence limits probe length and subsequent target specificity, and (ii) detection sensitivity, as short probes require significant amplification techniques to produce a visible signal; and finally (iii) small microRNA targets may be less stable in tissues (increased ribonuclease sensitivity and / or limited cross-linking to cellular components).

[0003] MicroRNAs regulate protein expression primarily by inhibiting translation and / or promoting mRNA degradation (Lin He and Gregory J. Hannon Nat. Rev. Genetics 5, 522-531). It is estimated that over 50% of mRNAs are regulated by one or more miRNAs, and a single miRNA can regulate several to dozens of genes. miRNAs have been shown to function in many physiological and pathological processes. miRNAs can function as tumor suppressors or oncogenes (Esquela-Kerscher et al. Nat. Rev. Cancer 6, 259-269.).

[0004] There are several RNA detection technologies commercially available. Ventana Medical Systems has products for automated kappa (kappa DNP probe catalog number: 495-524) and lambda (lambda DNP probe catalog number: 664-693) light chain mRNA expression in FFPET with commercial detection chemistry (ISH iVIEW Blue Plus Detection Kit catalog number: 760-097). This technology does not use 2'-O-methyl RNA oligonucleotide probes or amplification (e.g., tyramide signal amplification). Advanced Cell Diagnostics (ACD) has a wide range of products for manual detection of mRNA with its RNAScope technology. Extensive amplification mediated by branched DNA technology after hybridization is used to amplify the signal sufficiently for detection. Both chromogenic in situ hybridization (CISH) and fluorescent in situ hybridization (FISH) detection after branched DNA amplification are proposed by ACD. This technique is primarily used manually, with limited partial automation provided by the Ventana Discovery instrument. A substantial limitation overcome by the technique disclosed herein is that it requires a minimum RNA target length of 300 bases; therefore, the ACD technique cannot detect microRNA targets. Exiqon offers locked nucleic acid oligonucleotide probes for microRNA detection; the technique is limited to probe design and synthesis and does not provide any specific manual or automated detection capabilities (chromogen or fluorophore expression or deposition). Summary of the Invention

[0005] This disclosure describes a novel, highly sensitive, fully automated assay for the in situ detection of all RNA species, including secreted factor messenger RNA, non-coding RNA, and microRNA, in tissues, particularly formalin-fixed paraffin-embedded tissues (FFPET). It has been discovered that 2'-O-methyl modified RNA oligonucleotides labeled with detectable moieties (e.g., haptens) can be used as probes for in situ hybridization to RNA target molecules. The probes have been found to have increased in situ binding affinity for target RNA molecules compared to DNA probes, RNA oligonucleotide probes, or riboprobes, thus providing increased assay sensitivity. Furthermore, it has been discovered that 2'-O-methyl modified RNA oligonucleotides have better nuclease resistance than RNA probes or even DNA probes in terms of tissue staining.

[0006] Thus, two advantages offered by the new technology are that 2'-O-methyl RNA oligonucleotide probes have a higher affinity for RNA targets than DNA or RNA probes, and the methyl group confers resistance to RNases and other ribonucleases. In a specific embodiment, probes for miRNA have been developed. In some embodiments, each miRNA probe is labeled with two haptens, either directly synthesized (DNP) or using amine modification followed by haptenization with NHS.

[0007] This technology aims to provide a robust and efficient tool to explore the differential expression of certain miRNAs in cancer cells or in the tumor microenvironment in their surrounding normal tissues, which can be used as an effective tool for cancer diagnosis and treatment.

[0008] In this disclosure, a novel RNA detection technology, including modified RNA oligonucleotide probes designed for the detection of longer RNA targets and highly sensitive tyramide-hapten / silver detection, was employed for the automated detection of microRNA targets in formalin-fixed, paraffin-embedded (FFPE) tissues.

[0009] In an illustrative embodiment, a method for detecting target RNA in a tissue sample is disclosed. The method includes contacting the sample with an antigen retrieval reagent; contacting the sample with a labeled synthetic 2'-O-methyl oligonucleotide probe under conditions sufficient for the probe to hybridize to the target RNA in the sample; rinsing the sample to remove unbound probe; contacting the sample with an amplification reagent to deposit a plurality of amplification labels in the vicinity of the target RNA; contacting the sample with a detection reagent to deposit a detectable label in the vicinity of the target RNA; and detecting the target RNA by visualizing the detectable label. In one embodiment, the labeled synthetic 2'-O-methyl oligonucleotide probe is about 20 to about 200, about 20 to about 100 nucleotides in length. In another embodiment, the method uses conditions that preserve cell morphology.

[0010] Also disclosed are kits that include one or more of the probes or probe sets disclosed herein. Optionally, the kits can include additional reagents, such as signal amplification reagents (e.g., reactive chromogen conjugate-based reagents).

[0011] The methods of the invention may allow for the detection of more than one (e.g., 2, 3, 4, etc.) different targets. In some embodiments, a different detectable label and / or detection system may be used for each of the targets such that each may be individually detected in a single sample. Any suitable detectable label and / or detection system may be used.

[0012] More specifically, the invention features a system for bright-field in situ hybridization. In some embodiments, the system includes a probe set that includes X unique 2'-O-methyl RNA probes specific to a target RNA, where X > 2 (e.g., X = 2, X = 3, X = 4, X = 5, etc.), and the probes target X distinct portions within the target RNA. Each 2'-O-methyl RNA probe can be conjugated to at least one detectable moiety. The detectable moiety can be adapted to bind to a reactive chromogen conjugate system (e.g., a tyramide chromogen conjugate system) for signal amplification. In some embodiments, the 2'-O-methyl RNA probes each include a length of 15 to 30 nucleotides, 20 to 50 nucleotides, 40 to 80 nucleotides, 20 to 100 nucleotides, or 20 to 200 nucleotides.

[0013] In some embodiments, each of the 2'-O-methyl RNA probes is conjugated to at least two detectable moieties, at least three detectable moieties, at least four detectable moieties, or at least five detectable moieties. In some embodiments, the detectable moieties comprise a hapten. In some embodiments, the hapten comprises dinitrophenol (DNP). In some embodiments, the reactive chromogen conjugate system comprises a tyramide-hapten conjugate, or other suitable conjugate system. In some embodiments, each of the probes comprises at least one detectable moiety per 20 base pairs of the probe.

[0014] In some embodiments, the system further comprises a means for visualizing the target microRNA. In some embodiments, the means for visualizing the target microRNA comprises contacting the probe with a reactive chromogen conjugate system specific to the detectable moiety of the probe, the reactive chromogen conjugate system producing a color. In some embodiments, the system further comprises a means for visualizing the target microRNA, where the detectable moiety is visualized by the reactive chromogen conjugate system, and the visibility of the detectable moiety indicates the target microRNA. In some embodiments, the means for visualizing the target microRNA comprises a bright field microscope.

[0015] The invention also features a system for bright-field in situ detection of a microRNA target. In some embodiments, the system includes a target probe comprising a unique 2'-O-methyl RNA probe specific for a target microRNA, the 2'-O-methyl RNA probe being conjugated to at least one detectable moiety located at either the 3' or 5' end of the probe; and a reactive chromogen conjugate system effective for signal amplification, the reactive chromogen conjugate system being adapted to bind to the detectable moiety of the target probe.

[0016] In some embodiments, the 2'-O-methyl RNA probe comprises 15 to 30 nucleotides. In some embodiments, the probe comprises a first detectable moiety located at the 3' end of the probe and a second detectable moiety located at the 5' end of the probe. In some embodiments, the detectable moiety comprises a hapten. In some embodiments, the hapten comprises dinitrophenol (DNP). In some embodiments, the reactive chromogen conjugate system comprises a tyramide-hapten conjugate.

[0017] In some embodiments, the system further comprises a means for visualizing the target microRNA. In some embodiments, the means for visualizing the target microRNA comprises contacting the probe with a reactive chromogen conjugate system specific to the detectable moiety of the probe, the reactive chromogen conjugate system producing a color. In some embodiments, the system further comprises a means for visualizing the target microRNA, the detectable moiety being visualized by the reactive chromogen conjugate system, the visibility of the detectable moiety being indicative of the target microRNA. In some embodiments, the means for visualizing the target microRNA comprises a bright field microscope.

[0018] The present invention also features slides containing a plurality of cells chromogenically stained for a target RNA, the slides being generated using the systems disclosed herein (e.g., a target probe comprising a unique 2'-O-methyl RNA probe specific for a target microRNA, a probe set of unique 2'-O-methyl RNA probes, etc.).

[0019] The invention also features bright-field in situ hybridization methods. In some embodiments, the methods include contacting a sample with an antigen retrieval reagent; contacting the sample with a probe or probe set disclosed herein under conditions sufficient for the probe to hybridize to the target RNA in the sample; rinsing the sample to remove unbound probe; and detecting the target RNA by visualizing a detectable moiety.

[0020] In some embodiments, the method includes the steps of: contacting the sample with a probe specific for the target RNA under conditions sufficient for the probe set to hybridize to the target RNA in the sample, the probe set comprising X unique 2'-O-methyl RNA probes, X > 2, the 2'-O-methyl RNA probes targeting X distinct portions within the target RNA, each 2'-O-methyl RNA probe conjugated to at least one hapten; contacting the sample with a first anti-hapten antibody conjugated to a first enzyme, wherein the first anti-hapten antibody hybridizes to the X unique RNA probes. contacting the sample with a reactive chromogen conjugate system (e.g., a tyramide-hapten conjugate or any other suitable conjugate) where a first enzyme of the first anti-hapten antibody binds to the first anti-hapten antibody of the reactive chromogen conjugate system (the tyramide hapten conjugate also binds to the tissue); contacting the sample with a second anti-hapten antibody conjugated to a second enzyme, where the second anti-hapten antibody is specific for the reactive chromogen conjugate system and the second enzyme catalyzes the visualization of the chromogen, where the visualization of the chromogen is indicative of the target RNA.

[0021] In some embodiments, the method includes the steps of contacting the sample with a 2'-O-methyl RNA probe specific for a target RNA under conditions sufficient for the 2'-O-methyl RNA probe to hybridize to a target RNA in the sample, where the 2'-O-methyl RNA probe is conjugated to at least one hapten and is 15 to 30 nucleotides in length; contacting the sample with a first anti-hapten antibody conjugated to a first enzyme, where the first anti-hapten antibody is specific for the 2'-O-methyl RNA probe. The method includes the steps of: contacting the sample with a reactive chromogen conjugate system (e.g., a tyramide-hapten conjugate or any other suitable conjugate), where a first enzyme of a first anti-hapten antibody binds to the first anti-hapten antibody of the reactive chromogen conjugate system; contacting the sample with a second anti-hapten antibody conjugated to a second enzyme, where the second anti-hapten antibody is specific for the reactive chromogen conjugate system, the second enzyme catalyzing the visualization of the chromogen, and the visualization of the chromogen being indicative of the target RNA.

[0022] In some embodiments, the 2'-O-methyl RNA probe is conjugated to two haptens. In some embodiments, the hapten comprises dinitrophenol (DNP). In some embodiments, a first hapten is located at the 3' end of the probe and a second hapten is located at the 5' end of the probe.

[0023] In some embodiments, the methods include fixation utilizing a 2+2 system as described in U.S. Patent Application Serial No. 13 / 372,040, filed February 13, 2012, assigned to Ventana Medical Systems, Inc., and published as US20120214195 ('195), the contents of which are incorporated herein by reference in their entirety.

[0024] See, for example, Figures 5-9 detailing the effect of fixation on staining of MCF-7 xenograft tissue. The data detailed in Figures 5-9 show the effect at room temperature and various other temperatures utilizing the 2+2 method described and claimed in the '195 patent application. Briefly, the general method disclosed in the '195 patent application proposes a method of aldehyde fixation, exemplified by formalin fixation of a tissue sample, which comprises applying, immersing or otherwise contacting the tissue sample with an aldehyde solution at a first temperature for a first period of time, and then raising the temperature of the tissue sample to a second temperature, higher than the first temperature, for a second period of time.

[0025] As described in detail in the '195 patent application and as applied herein, the second temperature of the tissue sample is generally higher than the first temperature. The increase in temperature of the tissue sample may include rapidly or suddenly increasing the temperature of the tissue sample to the second temperature. The increase in sample temperature is done to increase crosslinking while still preserving the underlying sample reactivity. Alternatively, the increase in temperature of the tissue sample to the second temperature may be achieved by immersing the tissue sample in a solution at the second temperature, which may be the same or a different aldehyde solution. The second temperature is typically above ambient temperature, more typically above about 22°C, even more typically from above about 22°C to at least about 50°C, even more typically from above about 22°C to about 45°C. The second period of time is effective to allow substantially complete crosslinking of the endogenous molecules and structures to occur. The second period of time may vary, but typically ranges from more than 15 minutes to at least about 5 hours, typically from about 1 hour to about 4 hours, and even more typically from about 2 hours to about 3 hours. The rate and method used for increasing the temperature is designed to achieve optimal preservation of biomolecules prone to degradation such as microRNA / RNA and post-translational modifications. See '195 patent application.

[0026] The first period of time may vary depending on the tissue thickness, but typically ranges from about 15 minutes to about 4 hours, more typically from greater than 15 minutes to about 3 hours, and even more typically from about 1 hour to about 2 hours, according to the ASCO CAP guidelines for up to 4 mm thickness. It is recognized that for thicker samples, the first period of time will be dominated by the rate of diffusion. The first temperature is at least -20°C to about 15°C, typically at least 0°C to about 15°C, more typically at least 0°C to about 10°C, and even more typically from about 3°C ​​to about 5°C.

[0027] One embodiment of the method disclosed in the '195 patent application includes applying a first aldehyde solution at a first temperature to a tissue sample and then applying a second aldehyde solution to the tissue sample. The second aldehyde solution may be different from the first aldehyde solution. For example, the solutions may be of different concentrations or the second aldehyde solution may contain a different aldehyde than the first aldehyde. The aldehyde is typically a lower alkyl aldehyde, such as formaldehyde, glutaraldehyde, or a combination thereof.

[0028] As described in the '195 application, the 2+2 method offers at least three improvements over previous existing methods. First, enzyme activity can be significantly reduced by infiltrating formalin into the tissue sections in a cold environment. Second, by rapidly increasing the temperature of the tissue sample to increase the rate of crosslinking, cellular components are "locked" into place more quickly than would be observed at room temperature. This combination makes this technology superior to existing methods and, for the first time, allows for the preservation of modification states in FFPE tissues. Third, it is a general method that is believed to be applicable to a wide range of modification states and enzymes. While other methods target a specific set of modification enzymes, this method quickly disables all modification enzymes, thus preserving the general cellular state even better than Gold's standard room temperature procedure. The teachings of the '195 patent are: i. The present invention is not limited to a specific set of biomolecules or biomolecules containing specific post-translational modifications, and therefore the method is considered to be a general method for preservation of any biomolecule or modification state. Thus, the present invention is capable of preserving a large amount of biomolecules and biomolecules containing specific post-translational modifications with high quality. (Paragraph 0021) It continues:

[0029] Therefore, it is believed that the same effect is possessed by the method of the present invention.

[0030] The foregoing and other features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0031] The following drawings are submitted in color:

[0032] [Figure 1] 1 shows an example of the structure of a 2'-O' methyl RNA oligonucleotide.

[0033] [Diagram 2] FIG. 1 shows a schematic diagram of a method for detecting a probe of the present invention.

[0034] [Diagram 3] 1 shows a flow chart depicting exemplary steps for using 2'-O-methyl RNA probes in an assay.

[0035] [Figure 4A-B] MCF-7 xenograft tissue is shown, Figure 4A is a sample stained using a scrambled probe as a control to establish background staining, Figure 4B shows staining with miR-200c probe. [Figure 4C-D]Figure 4C shows staining with miR-21 probe, and Figure 4D shows staining with miR-25 probe. miR-21 showed the darkest staining, followed by miR-200c, then miR-25. The scrambled probe did not produce substantial background. The results show that the probes and methods described herein can be used to semi-quantitatively detect miRNAs in FFPET.

[0036] [Diagram 5] To test the effect of fixation on staining, MCF-7 xenograft tissues were harvested and immediately placed in room temperature or cold 10% NBF (neutral buffered formalin) under various conditions. Figures 5-9 show micrographs of miRNA staining for (A) negative control scrambled probe, (B) miR-21 probe, and (C) miR-200c probe. Figure 5 shows fixation using room temperature 10% NBF for 24 hours. [Figure 6] FIG. 6 shows fixation using NBF at 4° C. for 2 hours followed by NBF at 45° C. for 2 hours. [Figure 7] FIG. 7 shows fixation using NBF at 4° C. for 4 hours followed by NBF at 45° C. for 2 hours. [Figure 8] FIG. 8 shows fixation using NBF at 4° C. for 6 hours followed by NBF at 45° C. for 2 hours. [Figure 9] Figure 9 shows fixation using NBF at 4 °C for 24 hours followed by NBF at 45 °C for 2 hours. From Figures 5-9, it can be seen that the best signal-to-noise ratio for these specific microRNAs can be seen for samples treated with either the "2+2" (Figure 6) or "4+2" (Figure 7) fixation methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] I. Terminology Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless clearly dictated otherwise. Similarly, the word "or" is intended to include "and" unless clearly dictated otherwise. "Comprising" means "including." Thus, "comprising A or B" means "including A" or "including B" or "including A and B."

[0038] Suitable methods and materials for the practice and / or testing of the disclosed embodiments are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can also be used. For example, general methodologies well known in the art to which this disclosure pertains are described in a variety of general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and 2000 Supplement); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, the disclosures of which are incorporated herein by reference in their entireties.

[0039] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0040] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0041] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0042] Antibody: A polypeptide that contains at least a light or heavy immunoglobulin variable region and specifically binds to an epitope of an antigen (e.g., CD79a protein). Antibodies include monoclonal antibodies, polyclonal antibodies, or antibody fragments. Antibodies may be conjugated or labeled with a detectable label such as an enzyme, hapten, or fluorophore.

[0043] Detectable label (or detectable moiety): A compound or composition that is directly or indirectly conjugated to another molecule (e.g., a nucleic acid probe) to facilitate detection of that molecule. Specific non-limiting examples of labels include fluorescent and fluorogenic moieties, chromogenic moieties, haptens, affinity tags, and radioisotopes. Labels can be directly detectable (e.g., optically detectable) or indirectly detectable (e.g., through interaction with one or more additional molecules that are in turn detectable). Exemplary labels in the context of the probes disclosed herein are described below. Methods for labeling nucleic acids and guidance in the selection of labels useful for various purposes are discussed, for example, in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987, including revised editions).

[0044] Detectable labels can include chromogenic, fluorescent, phosphorescent and / or luminescent molecules, catalysts (e.g., enzymes) that convert one substance to another (e.g., by converting a colorless substance to a colored substance or vice versa, or by producing a precipitate or increasing the turbidity of the sample) to produce a detectable signal, haptens that can be detected through antibody-hapten binding interactions using an additional detectably labeled antibody conjugate, and paramagnetic and magnetic molecules or materials. Specific examples of detectable labels include enzymes, such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase or β-glucuronidase; fluorophores, such as fluoresceins, luminophores, coumarins, BODIPY dyes, resorufins, and rhodamines (many additional examples of fluorescent molecules can be found in The Handbook - A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR); nanoparticles, such as quantum dots (U.S. Pat. Nos. 6,815,064, 6,682,596, and 6,649,138, each of which is incorporated herein by reference in its entirety); metal chelates, such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions such as Gd3+; and liposomes, such as liposomes containing an entrapped fluorescent molecule. Where the detectable label comprises an enzyme, a detectable substrate, such as a chromogen, fluorogenic compound, or luminogenic compound, is used in combination with the enzyme to generate a detectable signal (a wide variety of such compounds are commercially available, e.g., from Life Technologies, Carlsbad, Calif.).

[0045] Alternatively, enzymes can be used in metallographic detection schemes. In some examples, metallographic detection methods involve the use of an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate for the enzyme. The substrate is converted by the enzyme to a redox-active agent, which reduces the metal ion to form a detectable precipitate (see, e.g., U.S. Pat. Nos. 7,642,064 and 7,632,652, each of which is incorporated herein by reference). In other examples, metallographic detection methods involve the use of an oxido-reductase enzyme (e.g., horseradish peroxidase) with a water-soluble metal ion, an oxidizing agent, and a reducing agent to again form a detectable precipitate (see, e.g., U.S. Pat. No. 6,670,113, incorporated herein by reference in its entirety). A hapten is a small molecule that can be bound by an antibody. Exemplary haptens include dinitrophenyl (DNP), biotin, digoxigenin (DIG), and fluorescein. Further haptens include oxazole, pyrazole, thiazole, nitroaryl, benzofuran, triperpene, urea, thiourea, rotenoid, coumarin and cyclolignan haptens, such as those disclosed in U.S. Pat. No. 7,695,929, which is incorporated herein by reference in its entirety.

[0046] Hybridization: The formation of base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a double-stranded molecule. Hybridization conditions that produce a particular degree of stringency will vary depending on the nature of the hybridization method and the composition and length of the nucleic acid sequences to be hybridized. In general, the temperature of hybridization and the ionic strength (e.g., Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. The presence of chemicals that reduce hybridization (e.g., formamide) in the hybridization buffer will also determine stringency (Sadhu et al., J. Biosci. 6:817-821, 1984). Calculations for hybridization conditions to achieve a particular degree of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 11). Hybridization conditions for ISH are also reviewed in Landegent et al., Hum. Genet. 77:366-370, 1987; Lichter et al., Hum. Genet. 80:224-234, 1988; and Pinkel et al., Proc. Natl. Acad. Sci. USA 85:9138-9142, 1988.

[0047] In situ hybridization (ISH): A method to determine the presence or distribution of nucleic acids in a sample using hybridization of labeled nucleic acid probes to localize specific DNA or RNA sequences in a portion or section of tissue (in situ), or in the entire tissue if the tissue is small enough (e.g., plant species, Drosophila embryos) (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes, for example for use in medical diagnostics to assess chromosomal integrity and / or determine gene copy number in a sample. RNA ISH measures and localizes mRNA and other transcripts within tissue sections or whole mounts.

[0048] In ISH, sample cells and tissues are usually treated to fix target nucleic acids in place and increase the accessibility of the probe to the target molecule. Detectably labeled probes hybridize to the target sequence at elevated temperature, and then excess probe is washed away. Solution parameters, such as temperature, salt and / or detergent concentration, can be manipulated to remove any non-identical interactions (e.g., so that only exact sequence matches will remain bound). The labeled probes are then localized and potentially quantified in the tissue, using either autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, which are typically differently labeled to simultaneously detect two or more nucleic acids.

[0049] Isolated: An "isolated" biological component (e.g., a nucleic acid molecule, a protein, or a cell) has been substantially separated or purified from other biological components in the preparation in which it occurs, the cells of an organism, or the organism itself, such as, for example, other chromosomal and extrachromosomal DNA and RNA, proteins and cells. "Isolated" nucleic acid molecules and proteins include nucleic acid molecules and proteins that have been purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins. In some examples, the nucleic acid probes disclosed herein are isolated nucleic acid probes.

[0050] Probe: A nucleic acid molecule that can hybridize to a target nucleic acid molecule (e.g., a genomic target nucleic acid molecule) and that, once hybridized to the target, can be detected, either directly or indirectly. Thus, a probe allows for the detection and, in some cases, quantification, of a target nucleic acid molecule. In certain cases, a probe comprises at least two segments that are complementary to a uniquely specific nucleic acid sequence of the target nucleic acid molecule, and thus can specifically hybridize to at least a portion of the target nucleic acid molecule. In general, once at least one segment or a portion of a segment has hybridized (and remains) to a target nucleic acid molecule, other portions of the probe may (but need not) be physically constrained from hybridizing to their cognate binding sites in other portions of the target (e.g., such other portions are too far away from their cognate binding sites); however, other nucleic acid molecules present in the probe can bind to each other, thus amplifying the signal from the probe. A probe may be referred to as a "labeled nucleic acid probe," indicating that the probe is directly or indirectly attached to a detectable moiety or "label" that allows the probe to be detected.

[0051] Sample: A specimen containing DNA (e.g., genomic DNA), RNA (including mRNA), protein, or a combination thereof obtained from a subject. Examples include, but are not limited to, chromosomal preparations, peripheral blood, urine, saliva, tissue biopsies, fine needle aspirates, surgical specimens, bone marrow, amniocentesis samples, and autopsy material. In one example, the sample contains genomic DNA. In some examples, the sample is a cytogenetic preparation, which may be placed, for example, on a microscope slide. In certain examples, the sample may be used directly or may be manipulated prior to use, for example, by fixation (e.g., using formalin).

[0052] Sequence identity: The identity (or similarity) between two or more nucleic acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured by percent identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured by percent similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.

[0053] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene, 73:237-44, 1988; Higgins and Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8:155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, Altschul et al., J. Mol. Biol. 215:403-10, 1990, provides a detailed discussion of sequence alignment methods and homology calculations.

[0054] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology Information and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Further information can be found on the NCBI website.

[0055] BLASTN can be used to compare nucleic acid sequences, while BLASTP can be used to compare amino acid sequences. If the two compared sequences have homology, the designated output file will present the regions of homology as aligned sequences. If the two compared sequences do not have homology, the designated output file will not present aligned sequences.

[0056] BLAST-like alignment tools (BLAT) may also be used to compare nucleic acid sequences (Kent, Genome Res. 12:656-664, 2002). BLAT is available from several sources, including Kent Informatics (Santa Cruz, Calif.) and the Internet (genome.ucsc.edu).

[0057] Once aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues are represented in both sequences. Percent sequence identity is determined by dividing the number of matches by either the length of the sequence defined in the specified sequence or by a segmented length (e.g., 100 consecutive nucleotides or amino acid residues from the sequence defined in the specified sequence) and then multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Percent sequence identity values ​​are rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded to 75.2. The length value will always be an integer. In another example, a target sequence that includes a 20 nucleotide region that aligns with 15 contiguous nucleotides from a sequence specified as follows contains a region that has 75 percent sequence identity to the specified sequence (i.e., 15÷20*100=75).

[0058] Subject: Any multi-cellular vertebrate organism, such as a human or non-human mammal (eg, a veterinary subject).

[0059] Target nucleic acid sequence or molecule: A defined region or specific portion of a nucleic acid molecule, such as a portion of a genome (e.g., a region of mammalian genomic DNA that contains a gene or gene of interest). In examples where the target nucleic acid sequence is a target genomic sequence, such a target can be defined by its location on a chromosome (e.g., in a normal cell), for example, by reference to a specific location on a chromosome according to cytogenetic nomenclature; by reference to its location on a genetic map; by reference to a virtual or assembled contig; by its specific sequence or function; by its gene or protein name; or by any other means that uniquely identifies it among other gene sequences of the genome. In some examples, the target nucleic acid sequence is a mammalian genomic sequence (e.g., a human genomic sequence).

[0060] In some instances, alterations in a target nucleic acid sequence (e.g., a genomic nucleic acid sequence) are "associated" with a disease or condition. In some instances, detection of the target nucleic acid sequence can be used to infer the state of a sample with respect to a disease or condition. For example, a target nucleic acid sequence can exist in two (or more) distinguishable forms, where a first form correlates with the absence of a disease or condition and a second (or different) form correlates with the presence of a disease or condition. The two different forms can be qualitatively distinguished, e.g., by a polynucleotide polymorphism, and / or the two different forms can be quantitatively distinguished, e.g., by the number of copies of the target nucleic acid sequence present in a cell.

[0061] Uniquely specific sequence: A nucleic acid sequence that is present only once in the haploid genome of an organism (e.g., a sequence of at least 20 bp (e.g., at least 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp or more)). In particular examples, a uniquely specific nucleic acid sequence is a nucleic acid sequence from a target nucleic acid that has 100% sequence identity with the target nucleic acid and does not have significant identity to any other nucleic acid sequences present in the specific haploid genome that contains the target nucleic acid.

[0062] Vector: Any nucleic acid that serves as a carrier for other (foreign) nucleic acid sequences that are not native to the vector. When introduced into a suitable host cell, the vector can replicate itself (and thus the foreign nucleic acid sequence) or express at least a portion of the foreign nucleic acid sequence. In one context, a vector is a linear or circular nucleic acid into which a nucleic acid sequence of interest is introduced (e.g., cloned) for purposes of replication (e.g., production) and / or manipulation using standard recombinant nucleic acid techniques (e.g., restriction enzyme digestion). A vector may contain nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. Common vectors include, for example, plasmids, cosmids, phages, phagemids, artificial chromosomes (e.g., BAC, PAC, HAC, YAC), and hybrids that incorporate features of more than one of these types of vectors. Typically, a vector contains one or more unique restriction sites (and in some cases multiple cloning sites) that facilitate the insertion of a target nucleic acid sequence.

[0063] II. Detailed probe design: 2'-O-methyl RNA oligonucleotides (2'OMe) have several desirable properties as probes for in situ detection of any RNA target in FFPE tissues: 1) 2'OMe probes have a higher affinity for RNA than DNA or RNA probes; 2) 2'OMe probes are resistant to nucleases; 3) the coupling efficiency during chemical synthesis is higher than DNA, resulting in a higher yield of full-length product. In FIG. 1 below, an exemplary structure of the 2'-O-methyl RNA oligonucleotide described herein is shown. For mRNA and non-coding RNA targets, 80 nucleotide (nt) long 2'OMe probes were selected to balance the number of haptens per probe and specificity. Each 80 nt 2'OMe probe contains five haptens by introducing one hapten every 20 nucleotides by chemical synthesis. Although a single 2'OMe probe can detect an RNA target, we experimentally determined that using a mixture of three independent 2'OMe probes for one RNA target further increases detection sensitivity without sacrificing specificity. For each RNA target, six 2'OMe probes were selected and attempted to be synthesized. By selecting the best three of the six synthesized, low yields, low sequence specificity, or other unforeseen problems could be mitigated. It was found that for most targets, any three of the six probes could be used interchangeably. For some RNA targets, only two or three 80 nt probes were selected.

[0064] To effectively select a set of six probes for mRNA or non-coding RNA targets, a bioinformatics toolset was constructed to semi / automate the entire process. The bioinformatics tool takes as one of the inputs either a sequence file or a GenBank ID; in the latter case, the software retrieves the sequences directly from GenBank. The tool then performs an iterative database search to mask any repetitive sequences as these sequences are highly abundant in the genome. Without masking the repeats, the resulting probes would generate unwanted background (lack of specificity). The tool then generated all potential probes with a predefined GC content and melting temperature range that avoids masking by the iterative database search. The tool was also used to take in other inputs such as ranges in the sequence and output format in case a specific region of RNA is targeted, etc. Again, the tool took all generated probes and performed a special BLASTN search designed for short oligonucleotides against the local genome, mRNA, and EST databases of interest, and selected or discarded probes based on the number of hits and associated BLAST bit scores and e-values ​​("NSE"). The remaining sequences were subjected to folding structure prediction analysis. The probes were folded with RNAfold, a leading open-source RNA molecule folding software, and probes with hairpin structures or low ΔG (cutoff -25 kcal / mol) were eliminated from further consideration. Six probes were selected for chemical synthesis from the remaining pool.

[0065] 2'-O-methyl RNA oligonucleotides were synthesized using a MerMade model 192 oligonucleotide synthesizer. A set of 30 probes of 80 nt length took approximately 48 hours to complete. The scale of synthesis was typically 50 nM, which produces enough probes to stain approximately 5 million slides of FFPET at the concentrations described here.

[0066] The quality of the synthesized 2'-O-methyl RNA probe was one of the determining factors for a successful ISH assay. Several procedures were found to improve the quality of the 2'OMe oligonucleotide probes from the synthesizer: 1) purification through a G50 column; 2) spectral measurements to determine if the number of haptens introduced was consistent with the design; and 3) gel electrophoresis to analyze the size distribution of the probes. The yield of G50 column purification of 80 nt 2'OMe probes ranged from 50-90%, with an average of about 65%. A successful probe contains + / - 10% of the expected hapten loading, for example for an 80 nt 2'OMe probe with 5 DNPs, the calculated number based on spectral measurements should be in the range of 4.5-5.5 DNPs per probe molecule.

[0067] In some embodiments, the 2'OMe probe has 100% sequence identity to the target RNA sequence. In some embodiments, the 2'OMe probe has between 99%-100% sequence identity to the target RNA sequence. In some embodiments, the 2'OMe probe has 95%-100% sequence identity to the target RNA sequence. In some embodiments, the 2'OMe probe has 90%-100% sequence identity to the target RNA sequence. In some embodiments, the 2'OMe probe has 80%-100% sequence identity to the target RNA sequence. In some embodiments, the 2'OMe probe has 75%-100% sequence identity to the target RNA sequence.

[0068] The invention also features a means for visualizing a target RNA. In some embodiments, the means for visualizing a target RNA includes contacting the probe with a detection reagent specific for the probe. Detection reagents are well known in the art. For example, the detection reagent can include an antibody or other probe that binds to the control probe. The detection reagent can include a molecule (e.g., an enzyme, a substrate, a tag) that visualizes a first label on the probe. The detection reagent can include multiple reagents (e.g., more than one antibody, enzyme, substrate, chromogen, etc.) effective to visualize the probe. In some embodiments, the detection reagent produces a color. Additional detection reagents (labels, tags, enzymes, substrates, chromogens, antibodies, etc.) are further disclosed herein. The invention also features a means for visualizing a target RNA, where the probe (e.g., a detectable moiety) is visualized by a detection reagent, and the visibility of the detectable moiety is indicative of the target RNA. Means for visualizing labeled probes are well known to those of skill in the art. For example, in some embodiments, the means for visualizing the target RNA comprises a microscope (e.g., a bright field microscope, a fluorescent microscope, an inverted microscope). In some embodiments, the means for visualizing the target RNA comprises a luminometer. In some embodiments, the means for visualizing the target RNA comprises a radiation detector (e.g., a gamma counter, etc.). In some embodiments, the means for visualizing the target RNA comprises a spectrometer. In some embodiments, the means for visualizing the target RNA comprises a real-time PCR machine. In some embodiments, the means for visualizing the target RNA comprises a scintillation and / or luminescence counter. In some embodiments, the means for visualizing the target RNA comprises a colorimeter. Other means for visualizing the target RNA are known in the art.

[0069] While not wishing to be bound by any theory or mechanism, it is believed that the probes of the present invention may allow for a reduction in the probe concentration required for effective hybridization, e.g., less probe is required for hybridization and sufficient signal, and thus the system of the present invention may help reduce the cost of the assay.

[0070] The disclosed methods can be automated. Systems for automated IHC and / or ISH are commercially available, such as the BENCHMARK ULTRA slide staining system, the BENCHMARK XT slide staining system, and the DISCOVERY XT slide staining system (Ventana Medical Systems, Tucson, AZ), the BOND-MAX and BOND-III slide stainers (Leica Biosystems, Buffalo Grove, IL), and the IQ Kinetic slide stainer (Biocare Medical, Concord, CA). Ventana Medical Systems, Inc. is the assignee of numerous U.S. patents that disclose systems and methods for performing automated analyses, including U.S. Patent Nos. 5,650,327; 5,654,200; 6,296,809; 6,352,861; 6,582,962; 6,827,901, and 6,943,029, each of which is incorporated herein by reference in its entirety.

[0071] Non-limiting examples of chromogens that can be used in the disclosed methods include (but are not limited to) those well known to those of skill in the art, such as those described in U.S. Patent Application Publication No. 2013 / 0260379 and U.S. Provisional Patent Application No. 61 / 831,552, filed June 5, 2013, both of which are incorporated herein by reference in their entireties.

[0072] The nucleic acid probes disclosed herein may include one or more labels, for example to enable detection of a target nucleic acid molecule. In various applications, such as in situ hybridization procedures, the nucleic acid probe includes a label (e.g., a detectable label). A "detectable label" is a molecule or material that can be used to generate a detectable signal that indicates the presence or concentration of a probe (e.g., a bound or hybridized probe) in a sample. Thus, the labeled nucleic acid molecule provides an indication of the presence or amount (e.g., gene copy number) of the target nucleic acid (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. Although specific examples are provided, the disclosure is not limited to the use of any particular label.

[0073] Labels associated with one or more nucleic acid molecules (e.g., the disclosed probes) can be detected directly or indirectly. Labels can be detected by any known or yet to be discovered mechanism, including absorption, emission, and / or scattering of photons (including photons of radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency). Detectable labels include colored, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (e.g., enzymes) that convert one substance into another resulting in a detectable difference (e.g., by converting a colorless substance into a colored substance or vice versa, or by creating a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

[0074] Specific examples of fluorescent molecules (or fluorescent dyes) are described herein. Numerous fluorescent dyes are known to those of skill in the art and can be selected, for example, from Life Technologies, see, for example, The Handbook - A Guide to Fluorescent Probes and Labeling Technologies. Examples of specific fluorophores that can be attached (e.g., chemically conjugated) to nucleic acid molecules (e.g., uniquely specific binding regions) are provided in U.S. Patent No. 5,866,366 to Nazarenko et al. (herein incorporated by reference in its entirety), including, for example, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene ... EDANS, 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, brilliant yellow, coumarin and derivatives such as coumarin and 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (7-amino-4-trifluorom ethylcouluarin)(coumarin 151);cyanosine;4',6-diaminidino-2-phenylindole (DAPI);5',5”-dibromopyrogallol-sulfonphthalein (bromopyrogallol red);7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaminepentaacetate;4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid Natostilbene-2,2'-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC);Eosin and derivatives, such as eosin and eosin isothiocyanate;Erythrosine and derivatives, such as erythrosine B and erythrosine isothiocyanate;Ethidium;Fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC ​​(XRITC); 2',7'-difluorofluorescein (OREGON) GREEN®); Fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-Methylumbelliferone; ortho-cresolphthalein; Nitrotyrosine; Pararosaniline; Phenol Red; B-Phycoerythrin; o-Phthaldialdehyde; Pyrene and derivatives such as pyrene, pyrene butyrate and 1-pyrenebutyrate succinimidyl; Reactive Red 4 (Cibacron Brilliant Red 3B-A); Rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxy Shirodamine (R6G), Lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates that emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, Lissamine; TM, diethylaminocoumarin, chlorotriazinyl fluorescein, naphthofluorescein, 4,7-dichlororhodamine and xanthene (described in U.S. Patent No. 5,800,996 to Lee et al., which is incorporated herein by reference in its entirety) and their derivatives, such as those available from Life Technologies (Carlsbad, Calif.), the ALEXA FLUOR® dye series (described, for example, in U.S. Patent Nos. 5,696,157, 6,130,101 and 6,716,979), the BODIPY dye series (dipyrrometheneboron difluoride dyes), and the like. Other fluorophores known to those skilled in the art can also be used, including, for example, U.S. Patent Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663, and 5,433,896, Cascade Blue (an amine-reactive derivative of sulfonated pyrene described in U.S. Patent No. 5,132,432), and Marina Blue (U.S. Patent No. 5,830,912), the disclosures of which are incorporated herein by reference in their entirety. In addition to the above fluorescent dyes, the fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, for example, a quantum dot (obtained, for example, from Life Technologies; see also U.S. Patent Nos. 6,815,064, 6,682,596, and 6,649,138, the disclosures of which are incorporated herein by reference in their entirety). Semiconductor nanocrystals are microscopic particles that have size-dependent optical and / or electrical properties. When semiconductor nanocrystals are irradiated with a primary energy source, a secondary emission of energy occurs at a frequency that corresponds to the band gap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light or fluorescence of a specific wavelength. Semiconductor nanocrystals with different spectral properties are described, for example, in US Pat. No. 6,602,671.Semiconductor nanocrystals can be attached to a variety of biomolecules (including dNTPs and / or nucleic acids) or substrates by techniques described, for example, in Bruchez et al., Science 281:2013-2016, 1998; Chan et al., Science 281:2016-2018, 1998; and U.S. Pat. No. 6,274,323, the disclosures of which are incorporated herein by reference in their entireties. The formation of semiconductor nanocrystals of various compositions has been disclosed, for example, in U.S. Pat. Nos. 6,927,069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and U.S. Patent Application Publication No. 2003 / 0165951 and PCT Publication No. 99 / 26299, the disclosures of which are incorporated herein by reference in their entireties. Separate populations of semiconductor nanocrystals can be produced that are distinguishable based on their different spectral properties. For example, semiconductor nanocrystals can be produced that emit different colors of light based on their composition, size, or size and composition. For example, quantum dots that emit at different wavelengths based on size (emission wavelengths of 565 nm, 655 nm, 705 nm, or 800 nm) are suitable as fluorescent labels in the probes disclosed herein and are available from Life Technologies (Carlsbad, Calif.).

[0075] Further labels include, for example, radioisotopes (such as 3H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions such as Gd3+, and liposomes.

[0076] Detectable labels that can be used with nucleic acid molecules (e.g., the disclosed probes) also include enzymes, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, or β-lactamase. Where the detectable label includes an enzyme, a chromogenic, fluorogenic, or luminogenic compound can be used in combination with the enzyme to generate a detectable signal (many such compounds are commercially available, e.g., from Life Technologies). Specific examples of chromogenic compounds include diaminobenzidine (DAB), 4-nitrophenyl phosphate (pNPP), fast red, fast blue, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, AP orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulfonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ON PG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, and tetrazolium violet.

[0077] Alternatively, enzymes can be used in metallographic detection schemes. For example, the silver in situ hybridization (SISH) procedure includes a metallographic detection scheme for identification and localization of hybridized genomic target nucleic acid sequences. Metallographic detection methods include the use of an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate for the enzyme. The substrate is converted by the enzyme into a redox-active agent, which reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005 / 0100976, PCT Publication No. 2005 / 003777, and U.S. Patent Application Publication No. 2004 / 0265922, the disclosures of which are incorporated herein by reference in their entirety.) Metallographic detection methods also include the use of an oxidoreductase enzyme (e.g., horseradish peroxidase) in combination with a water-soluble metal ion, an oxidizing agent, and a reducing agent, again forming a detectable precipitate. (See, for example, U.S. Patent No. 6,670,113).

[0078] In non-limiting examples, the disclosed nucleic acid probes are labeled with a hapten molecule (e.g., a nitroaromatic compound (e.g., 2,4-dinitrophenyl (DNP)), biotin, fluorescein, digoxigenin, and the like). Additional haptens suitable for labeling the disclosed probes include nitropyrazole, 3-hydroxyquinoxaline, thiazole sulfonamide, nitrocinnamic acid, rotenone, 7-(diethylamino)coumarin-3-carboxylic acid, benzodiazepine, or benzofuran haptens (see, e.g., WO 2012 / 003476, incorporated herein by reference). Methods for conjugating haptens and other labels to dNTPs (e.g., for ease of incorporation into a labeled probe) are well known in the art. For example procedures, see, e.g., U.S. Patent Nos. 5,258,507, 4,772,691, 5,328,824, and 4,711,955, the disclosures of which are incorporated herein by reference in their entireties. Indeed, many labeled dNTPs are commercially available, e.g., from Life Technologies (Carlsbad, Calif.). The label can be attached directly or indirectly to any position on the dNTP, e.g., to the phosphate (e.g., the α, β, or γ phosphate) or to the sugar.

[0079] Detection of the labeled nucleic acid molecule can be achieved by contacting the hapten-labeled nucleic acid molecule bound to the genomic target sequence with a primary anti-hapten antibody. In one example, the primary anti-hapten antibody (e.g., mouse anti-hapten antibody) is directly labeled with an enzyme. In another example, a secondary anti-antibody conjugated to an enzyme is used for signal amplification. In CISH, a chromogenic substrate is added, and in SISH, silver ions and other reagents as outlined in the referenced patents / applications are added.

[0080] In some instances, the probe is labeled by incorporating one or more labeled dNTPs using an enzymatic (polymerization) reaction. For example, the disclosed nucleic acid probes (e.g., incorporated into a plasmid vector) can be labeled by nick translation (e.g., using biotin, DNP, digoxigenin, etc.) or by random primer extension with terminal transferase (e.g., 3' tailing). In some instances, the nucleic acid probe is labeled by a modified nick translation reaction in which the ratio of DNA polymerase I to deoxyribonuclease I (DNase I) is modified to produce greater than 100% of the starting material. In particular examples, the nick translation reaction comprises DNA polymerase I to DNase I in a ratio of at least about 800:1, e.g., at least 2000:1, at least 4000:1, at least 8000:1, at least 10000:1, at least 12000:1, at least 16000:1, etc., e.g., about 800:1 to 24000:1, and the reaction is carried out at a substantially isothermal temperature, e.g., at about 16° C. to 25° C. (e.g., room temperature), overnight (e.g., about 16-22 hours). When the probes are included in a probe set (e.g., multiple plasmids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more plasmids), the plasmids can be mixed in an equimolar ratio prior to carrying out the labeling reaction (e.g., nick translation or modified nick translation).

[0081] In other examples, chemical labeling procedures can be used. Numerous reagents (including haptens, fluorophores, and other labeled nucleotides) and other kits are commercially available for enzymatic labeling of nucleic acids, including the disclosed nucleic acid probes. As will be apparent to those skilled in the art, any of the labeling and detection procedures disclosed above can be applied in terms of labeling probes, for example, for use in in situ hybridization reactions. For example, the MULTIPRIME® DNA labeling system from Amersham, various specific reagents and kits available from Molecular Probes / Life Technologies, or any other similar reagent or kit can be used to label the nucleic acids disclosed herein. In certain examples, the disclosed probes can be directly or indirectly labeled with haptens, ligands, fluorescent moieties (e.g., fluorophores or semiconductor nanocrystals), chromogenic moieties, or radioisotopes. For example, in indirect labeling, the label can be attached to the nucleic acid molecule via a linker (e.g., PEG or biotin). Further methods that can be used to label probe nucleic acid molecules are provided in US Patent Application Publication No. 2005 / 0158770, the disclosure of which is incorporated herein by reference in its entirety.

[0082] Further examples of haptens are described in US Pat. No. 8,846,320 and related patents (eg, US Pat. No. 7,695,929; US Provisional Application No. 60 / 856,133), the disclosures of which are incorporated herein by reference in their entireties.

[0083] While not wishing to limit the invention to any theory or mechanism, it is believed that the performance of the tyramide-hapten conjugates may be related to the hapten itself: for example, the hapten may affect (e.g., in an unexpected way) the formation of reactive tyramide-intermediates and / or the overall performance of the reagent.

[0084] While not wishing to limit the present invention to any theory or mechanism, it is believed that it is not always possible to predict which haptens will work best with a tyramide amplification system.

[0085] Because microRNAs are very short, it was anticipated that detection of the microRNA would require the use of a single probe. It was surprisingly discovered that a tyramide-hapten amplification system and '2-O-methyl RNA oligonucleotide probes are robust enough to detect microRNA targets.

[0086] III. RNA in situ hybridization and tyramide-chromogen detection details: Formalin-fixed paraffin-embedded tissues (FFPET) mounted on SUPERFROST slides were deparaffinized and antigen-retrieved using CC1 reagent and Protease 3 (Ventana Medical Systems, Inc. Cat#: 760-2020). After retrieval, one drop (100 μL) of hapten-labeled 2'O-methyl modified synthetic experimental antisense or control scrambled probe was dispensed onto the slide, denatured at 80°C for 8 min, and hybridized at 75°C for 2 h for 80 nucleotide probes against target RNA or at 45°C for 2 h for 25 nucleotide probes against miRNA (microRNA). After hybridization, slides were washed three times using EZ Prep (Ventana Medical Systems, Inc. Cat#: 950-102) at 75°C for 8 min for RNA 80 nucleotide probes or four times using 2X SSC at 45°C for 4 min for 25 nucleotide probes against miRNA to remove non-specifically hybridized probes.

[0087] It was found that sequential amplification is advantageous for detecting miRNA (e.g., chromogenically) in bright field microscopy. Tyramide-chromogen conjugates were used for detection. See US Patent Publication No. 2013 / 0260379, which is incorporated herein by reference for disclosure regarding detection chemistry. The use of a one-step amplification strategy involving an anti-hapten HRP conjugate to catalyze the deposition of the tyramide-chromogen conjugate did not produce a discernible signal. Here, in FIG. 2, a schematic diagram illustrating the detection approach is shown. In particular, the target 20 is detected by a probe 21 labeled with a hapten 22. An anti-hapten antibody 23 conjugated to an enzyme 24 is then contacted with the sample, such that the antibody 23 binds to the probe 21 and binds the enzyme 24 to the target 20. The enzyme 24 catalyzes the deposition of a tyramide-hapten conjugate 25. A plurality of tyramide-hapten conjugates 25 bind to the sample near the target 20, thus substantially amplifying the signal associated with the target 20. A second enzyme-conjugated anti-hapten antibody 26 is then contacted with the sample and allowed to bind to the tyramide-hapten conjugates 25. A second enzyme 27 can then be used to catalyze the deposition of a tyramide-chromogen conjugate, silver deposition (Ventana Medical Systems, Inc. Catalog #: 780-001), or any other chromogen desired. The final chromogen deposition is indicated as arrow 28, as the level of amplification compared to the signal 20 is very large.

[0088] 3, a flow chart showing exemplary steps for using 2'-O-methyl RNA probes in an assay is shown: deparaffinization of FFPET samples 31 is followed by recovery of RNA for detection 32 (e.g. mild CC1 or CC2), hybridization of probe to target 33, stringency washes 34, signal amplification 35, and chromogen detection 36.

[0089] Amplification and detection of each haptenylated probe was accomplished as follows: Endogenous tissue peroxidase activity was inactivated by dispensing one drop of PO inhibitor (Ventana Medical Systems, Inc. Catalog #: 760-4143) in triplicate and incubating the reaction for 12 minutes. After several washes, one drop of TSA block (Ventana Medical Systems, Inc. Catalog #: 760-4142) was dispensed onto the slide and incubated for 4 minutes, followed by one drop of HRP-conjugated anti-hapten monoclonal antibody (2.5ug / ml, prepared in Avidin Diluent plus B5 Blocker, Part # 90040); the mixture was incubated for 28 minutes.

[0090] Tyramide-mediated hapten amplification was achieved by dispensing one drop of tyramide-hapten conjugate onto a slide followed by one drop of TSA-H2O2 (Ventana Medical Systems, Inc. Catalog #: 760-4141) and incubating the reaction for 32 minutes. After extensive washing, another drop of TSA block was dispensed followed by one drop of cognate anti-hapten HRP-conjugated antibody that was incubated for 28 minutes. After washing, the HRP conjugate catalyzed silver dye deposition as indicated by the manufacturer (Ventana Medical Systems, Inc. Catalog #: 780-001). Tissue nuclei were then stained using hematoxylin II and bluing reagent; slides were then dehydrated using graded alcohols and coverslipped.

[0091] IV. Methods and Results The described automation technique has a modular design that is briefly described below.

[0092] 1) Treat formalin-fixed paraffin-embedded tissue (FFPET) samples on glass slides to remove wax, denature cross-linked proteins, and allow penetration of probes and detection reagents with limited amounts of proteases.

[0093] 2) A hapten-labeled probe is hybridized to the target in a hybridization buffer, followed by stringent washing to remove free probe.

[0094] 3) HRP-conjugated anti-hapten mouse monoclonal antibody is incubated with the hybridized probe, followed by extensive washing to remove any antibody that did not bind to the hapten on the probe.

[0095] 4) A tyramide amplification step is performed using a hapten-labeled tyramide compound and free tyramide is washed away.

[0096] 5) A second HRP-conjugated anti-hapten mouse monoclonal antibody is incubated with the hapten deposited by the tyramide reaction, and excess unbound antibody is washed away.

[0097] 6) In situ bound HRP catalyzes silver or alternative chromogen deposition and the signal is visualized under a microscope on the slide for detection and semi-quantification of RNA expression. EXAMPLES

[0098] Example 1 MicroRNA ISH staining concordant with array quantification To establish that the probes could be used to semi-quantitatively detect miRNAs in tissues with known concentrations, MCF-7 xenografts were analyzed. miRNA probes were selected based on a report by Fix et al. Cancer Genomics & Proteomics 7:261-278. The authors used microRNA microarrays to characterize and quantify 871 human miRNAs from MCF-7 cultured cells treated with or without Polyphenon-60. MCF-7 tissues were assayed for miR-200c (reported to have 10,000-20,000 copies per sample), miR-21 (reported to have >20,000 copies per sample), and miR-25 (reported to have 5,000-10,000 copies per sample).

[0099] DNP-labeled (5' and 3' ends) 22-nt 2'-O-methyl RNA oligonucleotide probes directed against four microRNA targets expressed at various levels in MCF-7 cells were synthesized and combined with a modified VENTANA OptiView amplification detection kit and silver detection; a fully automated staining assay was achieved using a VENTANA Benchmark XTR instrument. The probes and novel detection system catalyzed sensitive and specific detection of each microRNA target in MCF-7 xenograft FFPE tissue. The results are shown in Figure 4, where Figure 4A is a sample stained using a scrambled probe as a control to establish background staining. Figure 4B shows staining with miR-200c probe; Figure 4C shows staining with miR-21 probe; Figure 4D shows staining with miR-25 probe. Consistent with the published results, miR-21 showed the darkest staining, followed by miR-200c, then miR-25. The scrambled probe did not produce substantial background. The results show that miRNAs can be semi-quantitatively detected in FFPET using the probes and methods described here. Of note, detection can be amplified further or less than shown to alter the sensitivity of the staining.

[0100] A gradient of cytoplasmic microRNA signal was observed in normal fixed xenograft tissue (strong signal at the tissue periphery; weaker signal toward the tissue center), suggesting an effect of fixation on microRNA detection. A more uniform signal pattern was observed using "2+2" fixed MCF-7 xenograft tissue, suggesting that the utility of this novel fixation method may be extended to include microRNA targets.

[0101] To test the effect of fixation on staining, MCF-7 xenograft tissues were collected and placed immediately at room temperature or in cold 10% NBF under various conditions. Figures 5-9 show micrographs of miRNA staining for (A) a negative control scrambled probe, (B) a miR-21 probe, and (C) a miR-200c probe. Figure 5 shows fixation using room temperature 10% NBF (neutral buffered formalin) for 24 hours. The 24 hour room temperature slides are the desired standard against which processing protocols are compared to those disclosed in the '195 application. See Figure 5 of the '195 application. For purposes of this section addressing the effect of fixation on tissue staining, the general discussion disclosed in the '195 application is provided here.

[0102] As disclosed in the '195 patent application, the first step of the method is to subject the tissue sample to the fixative composition under conditions effective to allow substantially complete diffusion of the composition throughout substantially the entire cross-section of the sample. Effective temperature ranges for the first step are from greater than -20°C to at least 15°C, preferably from greater than 0°C to typically above about 10°C, and even more typically from about 3°C ​​to about 5°C. In the experimental examples, the temperature was typically about 4°C.

[0103] According to the teachings of the '195 patent application, increasing temperature increases the rate of cross-linking, and this initial processing step attempts to balance the beneficial properties associated with substantially complete diffusion of the fixative composition throughout the cross-section of the tissue sample while minimizing effects associated with initial cross-linking. However, because diffusion also increases with increasing temperature, it has been found that for a given sample, maximizing the rate of diffusion while minimizing any detrimental effects associated with increased cross-linking rates is likely to provide increased benefits.

[0104] The teachings continue that diffusion of the fixative composition into the tissue sample generally continues for a time effective to diffuse the composition throughout substantially the entire cross-section of the sample. The duration of the initial treatment step ranges from about 15 minutes to about 4 hours, most typically from greater than 15 minutes to about 3 hours, with good results typically being obtained by carrying out the fixative composition diffusion step for about 1.5 hours to about 2 hours.

[0105] The '195 patent application also teaches that the temperature associated with the second treatment step is typically above ambient temperature, e.g., above about 22°C. In the experimental examples described in that application, the temperature was typically above ambient temperature to at least 55°C, more typically from about 35°C to about 45°C, as this temperature range increased the crosslinking rate sufficiently to allow relatively rapid tissue crosslinking. The duration of the second treatment step ranged from greater than 15 minutes to at least about 5 hours, more typically from at least about 1 hour to about 4 hours, and more typically from about 2 hours to about 3 hours. One disclosed experimental example in the '195 application was conducted in which the second treatment step was at 45°C for 1.5 hours.

[0106] As an example, in the '195 patent application, Figures 4A and 4B therein are enlarged images of Figures 3G and 3H of the same application. The arrows therein for Figure 4B show the enlarged details of the results for images made using 2 hours pre-soaking in 10% formalin fixative at 4°C for these exemplary embodiments. Tissue morphology is shown to be better for tissue samples made using 2 hours pre-soaking in 10% formalin fixative at 4°C. In tissue samples that were not subjected to 2 hours pre-soaking in 10% formalin fixative at 4°C, formalin did not diffuse in - see the morphology of the tissue sample (Figure 4A) which is not as preserved as the morphology of the tissue sample made using 2 hours pre-soaking in 10% formalin fixative at 4°C (Figure 4B). The above is a brief description and teaching of the 2+2 method as described in detail in the '195 patent application and applied herein.

[0107] Figure 6 shows fixation using NBF at 4°C for 2 hours followed by NBF at 45°C for 2 hours; Figure 7 shows fixation using NBF at 4°C for 4 hours followed by NBF at 45°C for 2 hours; Figure 8 shows fixation using NBF at 4°C for 6 hours followed by NBF at 45°C for 2 hours; Figure 9 shows fixation using NBF at 4°C for 24 hours followed by NBF at 45°C for 2 hours. From Figures 5-9, it appears that the best signal-to-noise ratio for these particular microRNAs can be seen in samples processed with either the "2+2" (Figure 6) or "4+2" (Figure 7) fixation methods.

[0108] Equal

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0110] The technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," "containing," etc., should be read broadly and without limitation. In addition, the terms and expressions used herein are used as terms of description and not of limitation, and there is no intention to use such terms and expressions to exclude any equivalents or portions of the features shown and described, but it is recognized that various modifications are possible within the scope of the technology claimed.

[0111] Thus, it should be understood that the materials, methods, and examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the technology.

[0112] The present technology has been described broadly and generically herein. Each of the narrower species and subgeneric classifications falling within the generic disclosure also constitutes part of the present technology. This includes generic descriptions of the technology with any negative limitation or qualification that excludes any subject matter from the generic description, regardless of whether the excluded material is specifically described herein.

[0113] Additionally, when features or aspects of the technology are described in terms of Markush groups, those of skill in the art will recognize that the technology is also described in terms of any individual members or subgroups of members of the Markush group.

[0114] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0115] Other aspects are within the scope of the following claims.

Claims

1. 1. A system for bright field in situ detection of a first target RNA, comprising: at least one first target probe specific for at least a portion of a first target RNA, the at least one first target probe being conjugated to at least one first hapten, the at least one first target probe consisting of a 2'-O-methyl RNA oligonucleotide; a first reactive chromogen conjugate system effective for signal amplification, the first reactive chromogen conjugate system being adapted to bind to at least one first hapten of at least one first target probe, the first reactive chromogen conjugate system comprising a first tyramide-hapten conjugate system; A system including:

2. The system of claim 1 , wherein the first target RNA is a microRNA, a messenger RNA or a non-coding RNA.

3. 3. The system of claim 1 or 2, wherein at least one first target probe comprises a length of 15 to 30 nucleotides, a length of 20 to 50 nucleotides, a length of 40 to 80 nucleotides, a length of 20 to 100 nucleotides, or a length of 20 to 200 nucleotides.

4. The system of claim 1 , wherein the at least one first hapten is located at either the 3′ end or the 5′ end of the at least one first target probe.

5. The system of claim 1 , wherein at least one first target probe comprises at least two first haptens.

6. 2. The system of claim 1, wherein the at least one first hapten is selected from the group consisting of dinitrophenol (DNP), nitropyrazole, 3-hydroxyquinoxaline, thiazole sulfonamide, nitrocinnamic acid, rotenone, 7-(diethylamino)coumarin-3-carboxylic acid, benzodiazepines, and benzofurans.

7. The system of claim 1 , wherein the at least one first hapten is conjugated to the at least one first target probe via a linker.

8. The system of claim 1 , further comprising a means for detecting the first target RNA.

9. 9. The system of claim 8, wherein the means for detecting the first target RNA comprises contacting the probe with a first reactive chromogen conjugate system, the first reactive chromogen conjugate system emitting a detectable signal.

10. 10. The system of claim 1, further comprising a means for visualizing the first target RNA, wherein at least one first hapten is made visible by a first reactive chromogen conjugate system, and visibility of the detectable moiety is indicative of the first target RNA.

11. The system of claim 10 , wherein the means for visualizing the first target RNA comprises a bright field microscope.

12. The system of claim 1 , wherein the hapten is benzofuran.

13. The system of claim 1 , wherein the hapten is DNP.

14. The system of claim 1 , wherein the hapten is a nitropyrazole.

15. The system of claim 7 , wherein the linker comprises polyethylene glycol (PEG).

16. The system of claim 7 , wherein the linker comprises biotin.

17. 17. The system of claim 1, wherein the system comprises at least two target first probes, and each of the at least two target first probes is specific for a different portion of the first target RNA.

18. the system comprises at least one second target probe, the at least one second target probe being specific for at least a portion of a second target RNA, the at least one second target probe being conjugated to at least one second hapten, the at least one second target probe consisting of a 2'-O-methyl RNA oligonucleotide; and the system comprises a second reactive chromogen conjugate system effective for signal amplification, the second reactive chromogen conjugate system being adapted to bind to the at least one second hapten of the at least one second target probe, and the second reactive chromogen conjugate system comprising a second tyramide-hapten conjugate system.

18. A system according to any one of claims 1 to 17.

19. 20. The system of claim 18, wherein the at least one second hapten is different from the at least one first hapten.

20. 20. The system of claim 18 or 19, wherein the system comprises at least two second target probes, each of the at least two second target probes specific for a different portion of the second target RNA.

21. A slide comprising a plurality of cells stained for a first target RNA, the slide being prepared using the system of any one of claims 1 to 11.

22. contacting the sample with an antigen retrieval reagent; contacting the sample with at least one first target probe of the system of any one of claims 1 to 11 under conditions sufficient for the at least one first target probe to hybridize to a first target RNA in the sample; rinsing the sample to remove unbound first target probe; and Detecting the first target RNA by visualizing the detectable moiety. A method of bright-field in situ hybridization comprising:

23. 23. The method of claim 22, wherein conditions that preserve cell morphology are used.

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