Methods for diagnosing disease using multiplexed fluorescence and sequencing - Patents.com

Abstract

The present invention relates to methods for diagnosing disease by determining the presence or absence of mRNA species and / or at least one miRNA species of disease-associated biomarkers in a sample obtained from a subject via multiplex fluorescence in situ hybridization (FISH), and by determining the presence or absence of the mRNA species of the disease-associated biomarkers and / or the miRNA species of the disease-associated biomarkers of step (a) in the sample by multiplex sequencing. The present invention also relates to kits for carrying out the diagnostic methods described and provided herein, and to the use of such kits for carrying out the diagnostic methods described and provided herein.

Description

[Technical field]

[0001] The present invention relates to a method for diagnosing a disease by determining whether or not mRNA species of disease-associated biomarkers and / or at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten miRNA species are present in a sample obtained from a subject via multiplex fluorescent in situ hybridization (FISH) and by determining whether or not said mRNA species of disease-associated biomarkers and / or said miRNA species of disease-associated biomarkers of step (a) are present in said sample by multiplex sequencing.The present invention also relates to kits for carrying out the methods for diagnosis described and provided herein, and to the use of such kits for carrying out the methods for diagnosis described and provided herein. [Background technology]

[0002] Diagnosing diseases early and as reliably as possible is paramount to increasing the success of subsequent treatments. Biomarkers are becoming increasingly important for the diagnosis of all kinds of diseases, including biomarkers such as miRNAs (microRNAs). Appropriate FISH methods for detecting abundant biomarkers such as miRNAs have been previously described for the detection of skin cancer (Renwick et al., JCI (2013), 123(6): 2694-2702). However, such methods were developed for mostly abundant and cell type-specific nucleic acids such as miRNAs and were not easily used for other biomarker nucleic acids. Moreover, current FISH methods, including single molecule RNA FISH, do not guarantee accurate transcript abundance measurements, especially when only signal intensity is quantified and used for biomarker quantification.

[0003] Breast cancer (BCa) ranks first among all cancer types in women. One in eight women will develop BCa in their lifetime, with an estimated 550 000 deaths per year worldwide in 2017. While 44 000 women die from BCa in the United States, the situation is much worse in Europe, with 143 000 deaths. Around 571 000 new cases of invasive BCa were diagnosed in Europe in 2017. There are two major problems: 1. The current standard of care often does not allow for re-diagnosis; 2. Today's diagnostic tests lack accuracy and precision, resulting in false positive (20-30%), false negative (20-25%), and overdiagnosis (10-20%) results. Therefore, BCa patients are mis- / over-diagnosed and the resulting mis- / under- / over-treatment of approximately 280 000 BCa patients (approximately 50%) seriously contributes to a large number of BCa deaths in Europe, as well as increasing the social and economic burden on patients and society at large.

[0004] Breast cancer (BCa) is the most common cancer in women, with 1.7 million new cases diagnosed worldwide in 2012. This number is expected to double by 2030 due to aging, pollution, menopausal hormone use, lifestyle, obesity, etc. The incidence in Europe is about 600,000, affecting one in eight women. BCa is the second leading cause of cancer death in women. Today, almost 50% of BCa patients are misdiagnosed and later mis / under / overtreated, putting their lives at risk and in many cases causing death. To improve this rate, the diagnosis and treatment of BCa needs to be more precise and individualized. When a diagnosis of BCa is made, there are certain characteristics of the disease that oncologists need to know, such as tumor size, lymph node involvement, tumor grade, estrogen receptor status, progesterone receptor status, morphology, HER2 / neu status, etc. These characteristics create a picture of the cancer that is used to create a treatment plan specific to the patient. Often, this information is not clear enough to determine whether chemotherapy will be effective or not. Oncologists found that 46% of BCa patients did not benefit from chemotherapy. If those patients (whose BCa did not recur) received chemotherapy, the side effects and possible complications could outweigh the benefits of the drug. Obtaining 100% accurate BCa diagnostic data from a single diagnostic test that directly defines the most effective treatment, duration of treatment, and which patients will or will not benefit from chemotherapy would be beneficial for the diagnosis, prognosis, and treatment of BCa.

[0005] However, single tests provide only limited data. Conventional clinical protocols for diagnosing BCa (IHC, H&E, blood tests, etc.) are not fully quantitative (they cannot accurately measure the levels of BCa markers). In contrast, standard quantitative RNA-based methods (RT-qPCR, microarrays, etc.) are performed on samples isolated from tissues to measure the expression levels of BCa markers. However, they can only measure a few BCa markers and lack the histomorphometric information essential for the treatment of heterogeneous BCa. Therefore, oncologists must rely on a limited number of clinically validated biomarkers to select BCa treatments and perform several tests to obtain quantitative measurements and physical characteristics. Thus, conventional diagnosis results in 10-20% overdiagnosis in addition to 31% cancer misdiagnosis.

[0006] Moreover, there is an unreliable diagnostic rate (around 50%). Existing cancer profiling methods fail to reliably measure cancer markers in most cases. Testing of HER2 status performed on tissue sections using IHC can result in false positive / negative rates (>30%). False negative rates for ER / PR range from 20-25%, false positive rates for HER2 are 20-30%, and overdiagnosis is 10-20% (from both ISH and IHC). Misclassification of cancer leads to unnecessary toxic procedures and often causes death.

[0007] Also, the diagnosis is in most cases very long (>4 weeks). To achieve the most accurate diagnosis from current diagnostic tests, several tests must be performed (which is often not allowed by the current medical standards) and different laboratories are often involved. This results in a test turnaround time of >1 month and a high risk of false positive / negative rates. Other diagnostic solutions (Oncotype DX, MammaPrint, Prosigna) are slow to provide clinical information. Although it officially takes 2 weeks to receive the results, those diagnostic tests ultimately take longer than 4 weeks.

[0008] Furthermore, there are many cases of unnecessary chemotherapy (approximately 46%). With very limited BCa profiling and prognostic testing, many BCa patients receive unnecessary chemotherapy, and in 46% of BCa patients, chemotherapy is unnecessary. Summary of the Invention

[0009] There is a need to overcome these and further shortcomings. Therefore, the present invention addresses these needs and technical objectives and provides a solution as described herein and defined in the claims.

[0010] The present invention relates to a method for diagnosing a disease, the method comprising: (a) determining whether disease-associated biomarker mRNA species and / or at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten miRNA species are present in a sample obtained from the subject by multiplex fluorescent in situ hybridization (FISH); and (b) determining whether the mRNA species of the disease-associated biomarkers and / or the miRNA species of the disease-associated biomarkers of step (a) are present in the sample by multiplex sequencing. and preferably, that portion of the sample in which the mRNA species and / or at least one miRNA species of a disease-associated biomarker is determined to be present in step (a) is subjected to laser capturing prior to carrying out step (b).

[0011] The laser capture applied in the method of the present invention is preferably laser capture microdissection. Laser microdissection allows for example to isolate individual cells from a sample, particularly a tissue section sample, based on visual selection. Visual selection can be computer and software-assisted. Visual selection includes, among others, slide scanning, RNA visualization, RNA biomarker quantification, spectroscopic imaging, demixing, multiplex visualization, whole slide scanning, image analysis. Means and methods of laser capture microdissection are provided, for example, by Arcturus or Leica (see, for example, Hipp et al., J Path Inform 2018; 9, 45).

[0012] Preferably, signals, such as fluorescent signals, immunohistochemical signals, or signals from hematoxylin and / or eosin, can be detected by microscopy when multiplex FISH is performed. Such microscopy can be automated and computer and / or software assisted.

[0013] The inventors have observed that laser capturing significantly improves the results obtained by the method of the invention. In particular, the inventors have observed that laser capture microdissection reduces the bias introduced by sampling heterogeneous tissues and ensures concordance between RNA FISH and RNA sequencing (see FIG. 6). Thus, in the context of the method of the invention, it is preferred that, after performing multiplex RNA FISH, that portion of the sample in which the mRNA species and / or at least one miRNA species of disease-related biomarkers are determined by RNA FISH to be present is subjected to laser capturing, in particular laser capture microdissection, before performing multiplex RNA sequencing. Thereby, those portions of the sample that show a signal in the RNA FISH of one or more disease-related biomarkers are, so to speak, enriched and subjected to a potential confirmation step by performing multiplex RNA sequencing. In effect, it is determined whether the RNAs and / or miRNAs detected by multiplex RNA FISH can be (again) detected by multiplex RNA sequencing, thereby confirming the results obtained by multiplex RNA FISH. This two-step procedure avoids false positive and / or false negative results obtained, for example, by RNA FISH and / or RNA sequencing, since RNAs and / or miRNAs detected by multiplex RNA FISH need to be (again) detected by multiplex RNA sequencing.

[0014] In the context of carrying out the methods of the invention, particularly laser capture microdissection, it is preferred that the sample, particularly the tissue section sample, is placed on a glass slide, preferably on a positively charged glass slide, on a frame slide, on a frame slide with a PET membrane, or on a glass slide with a membrane.

[0015] Laser capture microdissection is preferably performed as described in the accompanying examples.

[0016] The preferred sample used in the method of the present invention is tissue section sample.Tissue section sample can be formaldehyde fixed, flash frozen (FFFF), or formaldehyde fixed (FF) and / or paraffin embedded (PE), preferably formaldehyde fixed, paraffin embedded (FFPE).Formaldehyde fixation is preferably carried out by formalin, which is 37% formaldehyde aqueous solution.

[0017] Preferably, the sample when used in the context of the present invention is obtained by microtome sectioning of a paraffin-embedded sample, which is preferably further formaldehyde-fixed. Ideally and preferably, the sample obtained by microtome sectioning is incubated at a temperature between 25°C and 0°C after acquisition. Indeed, the inventors have found it advantageous to place the sample, preferably the sample obtained by microtome sectioning after its acquisition, ideally immediately, at a temperature between 25°C and 0°C, for example 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C or 0°C, more preferably 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C or 0°C. Preferably, an ice-water bath may be used to incubate the samples at such temperatures.Microtome sectioning is preferably performed as described in the accompanying examples.

[0018] As shown in FIG. 4, when a sample, preferably a tissue section sample, preferably formaldehyde-fixed and paraffin-embedded, preferably obtained by microtome sectioning, after acquisition, is placed at a temperature of 25° C. to 0° C., which is achieved by an ice-cold water bath, the diffusion of RNA, particularly small RNAs, such as miRNAs, from the cells contained in the sample is inhibited, thereby ensuring a high concentration of RNA, particularly small RNAs. Indeed, FIG. 4 shows a FFFF tissue sample that serves as a baseline control. Although the sample, preferably processed as described herein, was placed at a temperature of 25° C. to 0° C. after its acquisition by microtome sectioning, the sample prepared according to the teachings of the prior art was not placed at such a low temperature. Indeed, the prior art does not teach placing a sample obtained by microtome sectioning at such a temperature. Rather, the prior art teaches immediately placing the sample at a temperature of about 45° C. to 42° C. in order to flatten the microtome sectioned sample.

[0019] If a sample that may be paraffin-embedded is to be diagnosed by the method of the invention, it is preferably deparaffinized before carrying out step (a) and / or step (b) of the method described herein. Deparaffinization is preferably achieved as described herein, in particular in the accompanying examples.

[0020] The present invention provides a novel personalized diagnostic test for several diseases, including but not limited to cancer (e.g., breast cancer (BCa)), that evaluates the expression levels of multiple biomarkers (mRNA species and / or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different miRNA species). It combines a visualization technique (FISH) followed by a multi-sample sequencing technique into a single test with a high level of specificity and sensitivity. This reduces analytical bias and allows for the simultaneous evaluation of multiple RNA biomarkers (e.g., 9-25 biomarkers in the case of BCa) on a single patient sample. The method of the present invention combines two independent qualitative / quantitative techniques, allowing cross-validation of biomarker analysis and eliminating or greatly reducing misdiagnosis. A specific personalized treatment and length of therapy can then be suggested, as well as the likelihood of recurrence, which helps oncologists design personalized and effective treatments. The methods of the invention described and provided herein can be applied to different diseases, particularly cancer, preferably any solid tumor (eg, breast cancer) and any stage of cancer.

[0021] As used herein, the terms "microRNA," "miRNA," or "miR," as defined herein, are used interchangeably and typically include non-coding RNAs of 18-26 nucleobases in length, which may be the product of cleavage of a pre-miRNA by the enzyme Dicer. Examples of mature miRNAs can be found in miRNA databases known in the art, such as miRBase (http: / / microma.sanger.ac.uk / ).

[0022] As used herein and known in the art, the term "mRNA" is used interchangeably with "messenger RNA" and refers to an RNA molecule that conveys genetic information from DNA to ribosomes, specifying the amino acid sequence of the protein product of gene expression. mRNA is unique to various genes and can show specific expression patterns in different types of tissues, such as tumor tissue.

[0023] Generally, as used herein, the terms "polynucleotide", "nucleic acid" or "nucleic acid molecule" should be interpreted synonymously. Generally, nucleic acid molecules can include, inter alia, DNA molecules (including cDNA, complementary DNA), RNA molecules (e.g., miRNA, mRNA, rRNA, tRNA, snRNA, siRNA, scRNA, snoRNA, and others known in the art), LNA (locked nucleic acid) molecules (see, e.g., Kaur et al., Biochem (2006), 45(23): 7347-7355), oligonucleotide thiophosphates, substituted ribo-oligonucleotides, or PNA (peptide nucleic acid) molecules. Furthermore, the term "nucleic acid molecule" can refer to DNA or RNA or hybrids thereof or any modifications thereof known in the art (see, e.g., US 5525711, US 471 1955, US 5792608, or EP 302175 for examples of modifications). Polynucleotide sequences may be single-stranded or double-stranded, linear or circular, natural or synthetic, and do not have any size limitations. For example, polynucleotide sequences may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribosomal RNA, or DNA that codes for such RNA or chimeraplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332-4339). Polynucleotide sequences may be in the form of vectors, plasmids, or in the form of viral DNA or RNA. Nucleic acid molecules that are complementary to the above nucleic acid molecules and that can hybridize to the nucleic acid molecules described herein are also described herein. The nucleic acid molecules described herein may be fragments of nucleic acid molecules in the context of the present invention. In particular, such fragments are functional fragments. An example of such functional fragments is a nucleic acid molecule that can serve as a primer.

[0024] As used herein, a nucleic acid molecule may contain different types of nucleotides, including naturally occurring nucleotides, modified nucleotides, and artificial nucleotides. As used herein, a nucleotide generally includes nucleosides, naturally occurring nucleosides, modified nucleosides, and artificial nucleosides. As known in the art, naturally occurring nucleosides contain purine or pyrimidine bases. Examples of naturally occurring nucleosides include (deoxy)adenosine, (deoxy)guanosine, (deoxy)uridine, thymidine, and (deoxy)cytidine. Nucleosides as part of the nucleotides (and thus nucleic acid molecules) described herein generally can include structures that contain any purine or pyrimidine nucleoside and its derivatives or analogs. That is, a "purine nucleoside" or a "pyrimidine nucleoside" used in the context of the present invention generally includes any type of purine or pyrimidine, and its derivatives or analogs, respectively, described herein, and a sugar, such as a pentose. In one embodiment of the invention, the purine nucleoside may be selected from the group consisting of (deoxy)adenosine, inosine, and (deoxy)guanosine, and derivatives or analogs thereof. The derivative may be, for example, a nucleoside having a purine selected from the group consisting of deazapurines, azidopurines, alkylpurines, thiopurines, bromopurines, O-alkylpurines, and isopurines, such as deazapurines, such as 7-deazapurines. That is, in one aspect of the invention, the purine nucleoside may be a nucleoside having a purine selected from the group consisting of deazapurines, azidopurines, alkylpurines, thiopurines, bromopurines, O-alkylpurines, and isopurines, such as deazapurines, such as 7-deazapurines. In another aspect of the invention, the purine nucleoside may be 1-methyl(deoxy)adenosine, 2-methyl-(deoxy)adenosine, N 6 -Methyl(deoxy)adenosine, N 6 ,N 6-Dimethyl(deoxy)adenosine, 7-deaza(deoxy)adenosine, 7-deaza-8-aza(deoxy)adenosine, 7-deaza-7-bromo(deoxy)adenosine, 7-deaza-7-iodo(deoxy)adenosine, 8-azido(deoxy)adenosine, 8-bromo(deoxy)adenosine, 8-iodo(deoxy)adenosine, 8-bromo-2'-deoxy(deoxy)adenosine, 2'-O-methyladenosine, inosine, 1-methylinosine, 2'-O-methylinosine, 1-methyl(deoxy)guanosine, 7-methyl(deoxy)guanosine, N 2 -Methyl(deoxy)guanosine, N 2 ,N 2 -Dimethyl-guanosine, isoguanosine, 7-deaza(deoxy)guanosine, 7-deaza-8-aza(deoxy)guanosine, 7-deaza-7-bromo(deoxy)guanosine, 7-deaza-7-iodo(deoxy)guanosine, 6-thio(deoxy)guanosine, O 6 In a further aspect of the invention, the purine nucleosides may be selected from the group consisting of queousine, archeusine, wyosine and N-methyl(deoxy)guanosine, 8-azido(deoxy)guanosine, 8-bromo(deoxy)guanosine, 8-iodo(deoxy)guanosine, 2'-O-methylguanosine, 8-azidoinosine, 7-azainosine, 8-bromoinosine, 8-iodoinosine, 1-methylinosine, and 4-methylinosine. 6-threonylcarbamoyl adenosine. In one aspect of the present invention, the pyrimidine nucleoside may be selected from the group consisting of (deoxy)cytidine, (deoxy)thymidine, (deoxy)ribothymidine, (deoxy)uridine, and derivatives thereof. The derivative may be, for example, a nucleoside having a pyrimidine selected from the group consisting of alkylpyrimidine, thiopyrimidine, bromopyrimidine, O-alkylpyrimidine, isopyrimidine, acetylpyrimidine hydropyrimidine, and pseudopyrimidine. That is, in one aspect of the present invention, the pyrimidine nucleoside may be a nucleoside having a pyrimidine selected from the group consisting of alkylpyrimidine, thiopyrimidine, bromopyrimidine, O-alkylpyrimidine, isopyrimidine, acetylpyrimidine hydropyrimidine, and pseudopyrimidine. In another aspect of the invention, the pyrimidine nucleoside is 3-methyl-(deoxy)cytidine, N 4 -Methyl(deoxy)cytidine, N 4 ,N 4 -Dimethyl(deoxy)cytidine, iso(deoxy)cytidine, pseudo(deoxy)cytidine, pseudoiso(deoxy)cytidine, 2-thio(deoxy)cytidine, N 4 -Acetyl(deoxy)cytidine, 3-methyl(deoxy)uridine, pseudo(deoxy)uridine, 1-methyl-pseudo(deoxy)uridine, 5,6-dihydro(deoxy)uridine, 2-thio(deoxy)uridine, 4-thio(deoxy)uridine, 5-bromodeoxy(deoxy)uridine, 2'-deoxyuridine, 4-thio(deoxy)thymidine, 5,6-dihydro(deoxy)thymidine, O 4-methylthymidine, difluorotoluene, and other nucleobase surrogates. As mentioned, the nucleosides described and provided herein generally include a purine or pyrimidine, or a derivative or analog thereof, as described herein, and a sugar moiety, such as a pentose. Generally, the pentose as part of the purine or pyrimidine nucleoside, or a derivative or analog thereof, as described herein, can be, inter alia, ribose, deoxyribose, arabinose, or methylribose (2-O-methylmethyribose), such as ribose or deoxyribose. That is, the nucleoside can be, for example, a (ribosyl)nucleoside, a desoxy(ribosyl)nucleoside, an arabinosylnucleoside, or a (methylribosyl)nucleoside, such as a (ribosyl)nucleoside or a deoxy(ribosyl)nucleoside.

[0025] As used herein, the terms "deoxy" and "deoxy" as prefixes to molecular terms are used interchangeably to indicate the absence of an oxygen atom or a hydroxyl group in a given pentose, such as, for example, ribose or other.

[0026] In the context of the present invention, the presence of disease-associated biomarkers in steps (a) and (b) is preferably indicative of the disease.

[0027] In a further aspect of the present invention, the methods described and provided herein allow for the diagnosis of disease by the presence or absence of a disease-associated biomarker.

[0028] Generally, the disease that is diagnosed by the method described and provided herein in connection with the present invention can be any disease that is linked to the presence of specific biomarkers that can be detected by FISH and can be sequenced.Therefore, in a preferred embodiment of the present invention, such biomarkers are nucleic acid molecules specific to each disease, preferably miRNA or mRNA, most preferably mRNA molecules.In one embodiment of the present invention, disease is cancer.In this context, cancer can be any type of cancer, for example, cancer is solid cancer. Specifically, when the disease is cancer, it can be, for example, a solid cancer (e.g., on-small cell lung cancer, breast cancer, colorectal cancer, pancreatic cancer, ovarian cancer, skin cancer, prostate cancer, cancer of the brain or nervous system, head and neck cancer, testicular cancer, lung cancer, liver cancer, kidney cancer, bladder cancer, gastrointestinal cancer, bone cancer, cancer of the endocrine system, cancer of the lymphatic system, fibrosarcoma, neuroectodermal tumor, mesothelioma, epidermoid carcinoma, or Kaposi's sarcoma), and a blood cancer (e.g., leukemia). In a particular embodiment of the invention, the disease is breast cancer (BCa).

[0029] According to the present invention, the mRNA species detected by the methods described and provided herein can be any mRNA whose elevated or decreased occurrence or expression is specific to a certain disease diagnosed as described herein. The elevated or decreased occurrence or expression can also be typical of a particular diseased tissue, such as tumor tissue (cancer). For example, in the case of breast cancer, typical mRNA species detected by the methods of the present invention include those specific to HER2 (HER2, GRB7), proliferation (Ki-67, STK15, survivin, cyclin B1, MYBL2), estrogen (ER, PR, Bcl2, SCUBE2), invasion (stromelysin 3, cathepsin L2), and others (GSTM1, BAG1, CD68), where HER2, ER, and PR represent the most relevant markers according to the present invention. Additional mRNA markers whose presence can be determined by the method of the present invention include HER2, ER, PR (the most relevant breast cancer diagnostic / prognostic transcript), NUPR1 (a marker for taxol resistance), and CSF1 (a marker for invasion in triple-negative breast cancer cases). Thus, in one embodiment of the present invention, additional mRNA species whose presence can be determined by the present invention can be selected from the group consisting of HER2, ER, PR, NUPR1, CSF1, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, ER, PR, Bcl2, SCUBE2, stromelysin 3, cathepsin L2, GSTM1, BAG1, and CD68, preferably including HER2, ER, and PR. Of course, in the method of the present invention, any mRNA that is a disease-related biomarker can be assayed for its elevated or decreased occurrence as described herein.

[0030] According to the present invention, at least one, two, three or more, for example, four, five, six, seven, eight, nine, ten or more miRNA species detected by the method described and provided herein can be any miRNA whose elevated or reduced occurrence or expression is specific to a certain disease diagnosed as described herein.The elevated or reduced occurrence or expression can also be typical of a certain diseased tissue, for example, tumor tissue (cancer).For example, in the case of breast cancer, the typical miRNA species detected by the method of the present invention include miR-21, miR-29a, miR-221 and miR-375. Thus, in one embodiment, the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten miRNA species whose presence may be detected by the present invention may be selected from the group consisting of miR-21, miR-29a, miR-221, and miR-375. Of course, in the methods of the present invention, any miRNA that is a disease-associated biomarker may be assayed for its elevated or decreased occurrence as described herein.

[0031] In one embodiment of the present invention, step (a) further comprises determining the presence or absence of snRNA (small nuclear RNA) and / or scRNA (small conditional RNA) species of disease-related biomarkers in the sample by multiplex FISH. According to the present invention, the snRNA or scRNA species detected by the method described and provided herein can be any snRNA or scRNA whose elevated or reduced occurrence or expression is specific to a certain disease diagnosed as described herein. The elevated or reduced occurrence or expression can also be typical (for cancer) of a particular diseased tissue, such as tumor tissue. For example, in the case of breast cancer, typical snRNA or scRNA species detected by the method of the present invention include U2 snRNA and 7SL scRNA (a novel biomarker pair for major breast cancer subtypes). Thus, in one embodiment, the snRNA or scRNA species whose presence can be determined by the present invention can be selected from the group consisting of U2 snRNA and 7SL scRNA. Of course, in the methods of the present invention, any snRNA or scRNA that is a disease-associated biomarker can be assayed for its elevated or decreased occurrence as described herein.

[0032] Furthermore, according to the present invention, the presence of a control marker can be determined by multiplex FISH in step (a). In one embodiment of the present invention, the presence of one or more additional RNAs selected from the group consisting of 28S rRNA, poly(A) RNA, β-actin, GAPDH, RPLPO, GUS, and TFRC can be determined by FISH as a control.

[0033] In a further embodiment of the invention, step (b) further comprises determining whether snRNA and / or scRNA species of the disease-associated biomarkers are present in the sample by multiplex sequencing. In a preferred embodiment in this context, and where step (a) further comprises determining whether snRNA (small nuclear RNA) and / or scRNA (small conditional RNA) species of the disease-associated biomarkers are present in the sample by multiplex FISH, step (b) further comprises determining whether snRNA and / or scRNA species of the disease-associated biomarkers are present in the sample by multiplex sequencing.

[0034] In the first step of the methods described and provided herein in connection with the present invention, multiplex fluorescent in situ hybridization (FISH) is used to determine whether mRNA species and / or at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten miRNA species of disease-related biomarkers are present in a sample obtained from a subject. As used herein, "multiplex FISH" refers to a FISH assay known to those skilled in the art (see, e.g., Lee et al., RNA (2011), 17(6): 1076-1089), in which the presence of mRNA species and / or at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten different miRNA species is determined. In one embodiment of the invention, the presence of several mRNA, scRNA, snRNA, rRNA, other RNA, and / or at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten different miRNA species is determined simultaneously by a "multiplex" FISH assay. According to the invention, simultaneous FISH measurement of different RNA species can result in multicolor FISH, since different RNA species can be detected using different dye labels.

[0035] Preferably, multiplex RNA FISH is performed as described in the accompanying examples.

[0036] For example, in one embodiment of the invention, particularly where the disease diagnosed by the method of the invention is breast cancer, the presence of at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten mRNAs (HER2, ER, PR) is determined simultaneously. In another embodiment of the invention, in addition to HER2, ER, and / or PR mRNA, the presence of (i) U2 snRNA and / or 7SL scRNA, (ii) 28SrRNA and / or poly(A) RNA (as a control and malignant tumor reporter), (iii) NUPR1 (a taxol resistance marker), and / or (iv) CSF1 (a marker of invasion in triple-negative breast cancer) is determined simultaneously by multiplex FISH.

[0037] In another embodiment of the invention, particularly when the disease diagnosed by the method of the invention is breast cancer, the presence of up to 25 biomarkers is determined simultaneously by multiplex FISH: mRNAs related to HER2 (HER2, GRB7) and / or mRNAs related to estrogen (ER, PR, Bcl2, SCUBE2), and one or more RNAs related to any of the following clusters: proliferation (Ki-67, STK15, survivin, cyclin B1, MYBL2), invasion (stromelysin 3, cathepsin L2), resistance (β-actin, GAPDH, RPLPO, GUS, TFRC), miRNAs (at least three of any one of miR-21, miR-29a, miR-221, or miR-375), and / or others (GSTM1, BAG1, CD68).

[0038] In general, the diagnostic methods described and provided herein in connection with the present invention are performed starting with a patient's tissue (e.g., cancer tissue, such as BCa tissue) removed during a biopsy or during surgery. The tissue is first analyzed by a multiplex FISH assay, allowing the simultaneous visualization and quantification of several (up to 9-25 for BCa) representative biomarkers. The assay is based on fluorescently labeled nucleic acid probes (e.g., available from MultiplexDX®) that recognize selected mRNA or miRNA targets. Hybridization of the FISH probe to the RNA allows visualization of the biomarkers in single cells by common fluorescence microscopy. In one embodiment of the invention, the invention may use "click labeling" to increase the binding affinity of FISH probes based on the chemical fusion of two adjacent probes on a specific RNA target (see FIG. 2). Thus, it is preferred that the probes used for fluorescent in situ hybridization (FISH) of RNA can be linked by click chemistry. Click chemistry cross-linking of the probes is preferably performed as described in the appended examples. According to the present invention, the specificity of the FISH probes allows for simultaneous (multiplexed FISH) detection of multiple biomarkers (multicolor FISH) and accurate biomarker quantification derived from FISH signal intensity.

[0039] As known to those skilled in the art, in FISH, any mishybridization to relatively stable abundant RNAs will result in a false positive signal that reduces specificity. Although probe hybridization in FISH assays cannot be directly compared to primer or siRNA hybridization in PCR or RNAi, respectively, it is clear that during PCR, the end of the primer, where perfect binding is required to initiate the enzymatic chain reaction, is most sensitive to mismatches or bulges in the primer-target duplex that would cause an aberrant PCR reaction. While 1% mishybridization of siRNA to abundant RNAs such as ribosomal RNA (rRNA) is irrelevant, such levels of mishybridization to abundant nonspecific transcripts in RNA FISH would result primarily in rRNA signal (since rRNA is at least 1000-100,000 times more abundant than mRNA, 1% mishybridization to rRNA would be up to 1,000 times higher than the target mRNA signal). Thus, probe mishybridization remains a major technical barrier.

[0040] The probe used in the method of the present invention described herein can be of any type suitable for application in FISH as known in the art.Preferably, the probe is based on a nucleic acid molecule.In one embodiment of the present invention, the probe is an LNA molecule.The probe can be of any length, depending on the RNA molecule whose presence is determined by the method of the present invention.For example, in one embodiment of the present invention, the probe can be up to 40 nucleotides long, preferably up to 35, 30, or 20 nucleotides long.In a further embodiment of the present invention, the probe is at least 7 nucleotides long, preferably at least 8, 9, or 10 nucleotides long.

[0041] Probes for RNA detection in FISH can be designed as generally known in the art. For example, according to the present invention, the following parameters are preset: 1. target sequence (RNA of interest), 2. hybridization temperature for the RNA FISH protocol, and 3. desired number of FISH probes per target. A list of sequences that can be subsequently inserted into the synthesis program of the DNA synthesizer can then be generated by the program provided by MultiplexDX® or other commercially available programs. Based on predicted hybridization behavior and minimal cross-hybridization with the most abundant RNAs (e.g., rRNA, ncRNA, tRNA, mt-RNA, scRNA, etc.), appropriate sequences can be selected for the probes. According to the present invention, optimal probes can satisfy the following characteristics: (a) minimized cross-hybridization with the most abundant RNAs, and (b) equalized LNA / DNA melting temperatures (T M ) / binding affinity, precise positioning of the probe, and LNA content per probe. Based on the selected hybridization conditions, the RNA FISH probes can be, for example, 11-15 nt long locked nucleic acid (LNA)-modified DNA probes (LNA / DNA) with about 3-5 LNA modifications per probe. M The predictions are based on algorithms currently used by, for example, IDT DNA oligo analyzers for DNA / DNA, DNA / RNA, and LNA / DNA duplexes, and take into account the target type (DNA or RNA), oligo concentration, and Na + Concentration can be used as the main input parameter, which can optionally be modified according to the invention to 50% formamide, which can be used as a denaturing agent in RNA FISH protocols.

[0042] For multiplex FISH used in connection with the methods of the invention, for example, short, fluorescent, multiply labeled LNA / DNA probes (about 11-15 nt long, about 4-8 fluorophores per probe, about 20-50 probes per target) can be used to visualize any RNA target (up to 5 kb). The probes can be synthesized, deprotected, and desalted, each with any desired modifications as needed, for example, on an automated Dr. Oligo 48 (see http: / / www.biolytic.com / t-dr-oligo-48-dna-rna-oligo-synthesizer.aspx) or Dr. Oligo 768 (see https: / / www.biolytic.com / t-dna-rna-oligo-synthesizer.aspx) DNA / RNA synthesizer or other means known in the art. Each fluorescent multi-labeled probe (e.g., about 11-15 nt) can be prepared with an extension segment containing multiple (4-8) 5-octadiynyl-dU or Abasic-Alkyne or other suitable dyes, each separated by, for example, a 5 nt segment or a C18 spacer (GlenResearch). According to the present invention, this allows very strong, selective, and efficient click chemistry labeling with azide-labeled fluorophores or any fluorophore of interest (e.g., ATTO dyes, Alexa dyes, Cy, BODIPY dyes, DyLight dyes, Cyto dyes, Seta dyes, RadiantDy dyes, and CF dyes, selecting some of the brightest, most photo-soluble and water-soluble, and avoiding non-specific protein binding). Click chemistry, due to its robust specificity and quantitative efficiency when compared to NHS-ester chemistry, is, according to the present invention, a suitable approach that allows the multi-labeling of probes with 5 or more fluorophores per probe.Comparing click chemistry labeling to NHS-ester labeling, fluorophore-azides are 33-50% more expensive than fluorophore-NHS esters, however, according to the present invention, only about, e.g., 1.2-fold excess of azide is needed instead of the usual 10-fold excess of NHS-ester in the oligo labeling reaction, making click chemistry more cost-effective.

[0043] According to the present invention, the multiplex FISH used in the method of the present invention described and provided herein can be carried out, for example, as follows. In one embodiment, tissue cells can be fixed before the detection of biomarkers described herein. Suitable methods for fixing cells and tissues and then detecting biomarkers are known in the art, and are described, for example, in US 9359636, US 9005893 and US 8394588. According to the present invention, the in situ click ligation of two adjacent probes (about 15 nt long) forms a more stable double probe of 30 nt long (see FIG. 2), followed by stringent washing targeted at mishybridization, which completely washes out the mishybridized probes while maintaining the complete specific binding of all probes, reducing background (see FIG. 2). This allows specific RNA FISH signals to be obtained even with low amounts of RNA targets.

[0044] As mentioned above, it is preferred that the sample is fixed before carrying out step (a) of the method described herein. Fixation can preferably be achieved by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 5-ethylthio-1H-tetrazole (ETT). Nevertheless, fixation is preferably carried out as described herein, in particular in the accompanying examples.

[0045] With regard to click labeling as described in the context of the present invention, for example, one probe may be labeled with azide (through a 3'-amino-modified LNA / DNA oligonucleotide) and the other may be tagged with an alkyne at the 5' end, directly labeled during oligonucleotide synthesis (see FIG. 2). Each probe may preferably be labeled with the same fluorophore. This approach has been tested in vitro as shown in FIG. 2. Click chemistry may then be performed at room temperature to bind the probes. This significantly increases the binding affinity of the formed double probe. This in turn may allow the use of stringent washes targeting mishybridization, whereby mishybridization from the 15 nt long probe is completely washed away by increasing the temperature of the wash above the previous hybridization temperature (e.g., if the hybridization temperature is 40°C, the stringent wash may be 45-50°C). The increased stability of the double probes after binding and stringent washes may maintain complete and specific binding of all probes. According to the present invention, a multiplex FISH approach can be performed as follows: 1. RNA FISH using 15 nt long LNA / DNA probes at 40°C; 2. Click chemistry performed at cooling and room temperature; 3. Stringent washing at 45-50°C (5-10°C higher than the previous hybridization temperature).

[0046] As described herein, multiplex FISH used in connection with the methods of the invention described and provided herein allows for the simultaneous determination of the presence of several RNA species described herein. For example, according to the present invention, 8-color multiplex FISH can be applied as follows. Figure 3 depicts 8-color multiplex FISH, allowing for the simultaneous detection of seven or more different fluorophores with partially overlapping spectra. Since the RNAs are abundant in different subcellular localizations (U1, U2 snRNA: nuclear, mt-RNA: mitochondrial, 7SL scRNA: cytoplasmic, U3 snoRNA: nucleolus, rRNA: nucleolus, poly(A): nuclear / cytoplasmic), the specificity of the signals can be visually determined (see the red rectangles showing the specific signals on one cell, Figure 3). Spectral imaging with linear unmixing can be used to check whether all individual signals in the entire spectrum (sum of all spectra) should be approximately equal, which is considered to be one of the most relevant limitations of current methods. The following parameters can be selected according to the present invention. 1. T M Specific probes with equalized intensity are additively detectable (signal intensity of 4 probes = 4 times the intensity of a single probe), which allows control of the number of probes per target (if the signal is too strong or too weak, the number of probes can be reduced or increased, respectively). 2. If an increase in the number of probes is not possible, dyes with higher quantum yields can be used; 3. If more powerful fluorophores are insufficient, twice the number of fluorophores per probe can be used. Furthermore, the use of directly labeled LNA / DNA probes without signal amplification allows accurate quantification of the signal intensity of each RNA target (see Table 1). The normalized relative RNA FISH intensity correlates perfectly with the RNA sequencing data, demonstrating the quantification potential of the method described and provided in connection with the present invention.

[0047] Table 1: Comparison of ncRNA small RNAseq sequencing counts and normalized intensities derived from RNA FISH. Read counts were determined using over 2000 small RNA cDNA libraries from various tissues. All probes were conjugated to the same ATTO-550 dye and intensity was measured using the same window size containing approximately 200 cells. A poly(T) probe targeting the poly(A) tail of mRNA was conjugated to ATTO-647N and used in combination with other probes, followed by poly(A) signal to normalize intensity as follows: Relative fluorescence intensity normalized to poly(A) = [measured intensity / (number of fluorophores in probe set] * exposure time)] / [measured intensity / (number of fluorophores in poly(A) probe * exposure time). TIFF0007618234000001.tif49161

[0048] According to the present invention, signal quantification can be achieved as exemplarily shown in Table 2, which shows eight LNA / DNA probes targeting mouse 28S rRNA and their intensities in mouse cerebral cortex sections. The probes are incubated at similar T temperatures at least 10-20°C higher than the hybridization temperature. M The eight probes were designed to have similar binding affinities as indicated by the binding affinity of the probes. Table 2 shows that all the intensities are nearly equal, and also demonstrates that pooling all eight probes results in a total signal intensity that is eight times higher than the signal intensity of the individual probes. The fluorescent signal intensity of all eight probes pooled together (498472) was only 1.2% higher than the sum of all signal intensities from the single probes (492126), indicating high accuracy. This linear increase is consistent with the linear increase seen when the probes have similar binding affinities and are not washed off during hybridization (the T of the probes). M is at least 10° C. higher than the hybridization temperature), indicating that the method of the present invention can be used for accurate target quantification.

[0049] Table 2: Mouse 28S rRNA probe sequences. Eight oligonucleotide probes were designed to detect mouse 28S rRNA in neurons of the mouse cerebral cortex using RNA FISH. LNA residues are shown in lowercase. Signal intensities were recorded for each probe individually, in combination (rRNA1-8), and in their absence (blank). The signal intensity of each probe was approximately 100-fold higher than that of the blank control. Pooling all eight probes yielded a signal intensity 8-fold higher than that of the individual probes, indicating that RNA FISH can be used for accurate target quantification (Σ[rRNA1,..,rRNA8]=492126). The melting temperatures (T M ) is shown. T was analyzed using 50% formamide, 1 M NaCl, 50 mM phosphate (pH 7.0). M All probes (100 nM) were hybridized for 16 h at 55° C. in hybridization buffer containing 50% FA and 1 M NaCl. N≧100 cells. TIFF0007618234000002.tif92161

[0050] The term "hybridization" or "hybridizing" as used herein in connection with nucleic acid molecules / sequences, including miR, mRNA, DNA, cDNA, LNA, and others described herein, or portions or fragments thereof, can refer to hybridization under stringent, low stringent, or non-stringent conditions. In one embodiment, the conditions are preferably stringent. Hybridization conditions can be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001); Current Protocols in Molecular Biology, Update May 9, 2012, Print ISSN: 1934-3639, Online ISSN: 1934-3647; Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY (1989), or Higgins and Hames (Eds.), "Nucleic acid hybridization, a practical approach", IRL Press Oxford, Washington DC, (1985). Setting conditions is well within the skill of a person of ordinary skill in the art and can be determined according to protocols described in the art. Therefore, stringent hybridization and washing conditions (as used herein, "stringent conditions"), such as 0.1×SSC, 0.1% SDS at 65° C., are generally considered necessary for the detection of only specifically hybridizing sequences. Non-stringent hybridization conditions for the detection of homologous or non-exactly complementary sequences may be set at 6×SSC, 1% SDS at 65° C. (as used herein, "non-stringent conditions").As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. The above conditions can be changed by the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary preparations. The inclusion of a particular blocking reagent may require modification of the above hybridization conditions due to compatibility issues. According to the invention described herein, low stringency hybridization conditions for the detection of homologous or non-exactly complementary sequences can be set, for example, at 65° C. with 6×SSC, 0.5% SDS (as used herein, “low stringency conditions”). As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.

[0051] The nucleic acid molecules that hybridize also include fragments of the above molecules. Such fragments may correspond to the nucleic acid molecules or functional fragments thereof that function as inhibitors as described herein. Furthermore, the nucleic acid molecules that hybridize to any of the above nucleic acid molecules also include complementary fragments, derivatives and variants of these molecules. In addition, hybridization complexes refer to complexes between two nucleic acid sequences (e.g., cDNA / LNA) by the formation of hydrogen bonds between complementary G and C bases and complementary A and T (or U in the case of RNA, as known to those skilled in the art): these hydrogen bonds can be further stabilized by base stacking interactions. Two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. Hybridization complexes can be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membrane, filter, chip, pin, or slide on which cells are immobilized). The term "complementary" or "complementarity" refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing. For example, the sequence "AGU" binds to the complementary sequence "UCA." Complementarity between two single-stranded molecules can be "partial," where only some of the nucleic acids bind, or complete, where there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions, which depend on binding between nucleic acid strands.

[0052] When performing RNA FISH as described herein, it is preferred to stain the sample with hematoxylin and eosin after performing RNA FISH.

[0053] As described herein, in the context of the method of the present invention, 28S rRNA and poly(A) RNA are determined by FISH as a control.This control, namely 28S rRNA and poly(A) RNA, is particularly interesting as a control, because the ratio of poly(A) RNA to 28S rRNA indicates possible RNA degradation in sample.Therefore, it is preferable to check the ratio of poly(A) (m)RNA to 28S rRNA before carrying out step (a) and / or step (b) of the method.

[0054] In fact, the inventors have observed that the quality control of samples described herein can be useful before performing multiplex RNA FISH and / or multiplex RNA sequencing.Surprisingly, the inventors have found that the effect on RNA degradation is reflected by the ratio of polyA (m) RNA / 28S rRNA.As shown in Figure 5, the ratio of polyA (m) RNA / 28S rRNA (fluorescence activity / unit) can be used to evaluate RNA degradation in samples, such as formalin-fixed paraffin-embedded (FFPE) tissue section samples, with a ratio of less than 1 signifying degraded RNA (the presumably ideal ratio of 1.15 is marked as a dotted line in Figure 5).

[0055] Therefore, prior to carrying out multiplex RNA FISH (step (a) referred to herein) and / or multiplex RNA sequencing (step (b) referred to herein) in the context of the method of the present invention, the samples described herein are preferably checked whether the ratio of polyA(m)RNA to 28S rRNA is 1:1 or greater than 1:1, e.g. 1.05:1, 1.1:1, 1.15:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1 or 3:1.

[0056] However, in an alternative, prior to carrying out multiplex RNA FISH (step (a) referred to herein) and / or multiplex RNA sequencing (step (b) referred to herein) in the context of the method of the present invention, the samples described herein are preferably checked whether the ratio of polyA(m)RNA to 28S rRNA is 1:1 or less than 1:1, e.g. 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:2 or 1:3.

[0057] As described herein, according to the method provided by the present invention, the multiplex FISH step is followed by a multiplex sequencing step (step (b)). Thus, after the FISH step, a sequencing step is performed to confirm the result of step (a) (multiplex FISH) and / or to determine the presence of additional biomarkers indicative of each disease. That is, in the context of the present invention, the same biomarkers whose presence was determined in step (a) can be sequenced in step (b), or other or additional biomarkers other than those in step (a) can be sequenced in step (b). In one embodiment of the present invention, the same biomarkers whose presence was determined in step (a) can be sequenced in step (b). The sequencing of biomarkers can be performed by methods known in the art and methods described and exemplified herein. For example, according to the present invention, after the tissue is stained in step (a) during the FISH assay and when the disease to be diagnosed is cancer (e.g., breast cancer), the tumor can be selectively isolated from normal tissues and immune cells, for example, by laser microdissection. Total RNA can then be extracted from the homogenous population of cancer cells and subjected to RNAseq (see Canovas et al., Sci Reports (2014), 4 (art. no. 5297), doi: 10.1038 / srep05297) to confirm the quantification of the first analysis (visualized with multiplex FISH in step (a)) and to reveal the presence and amount of the same or a wider panel of specific biomarkers. Thus, in the context of the method of the invention, it is preferred that RNA is extracted from the sample when multiplex sequencing of RNA is performed. Preferably, RNA extraction is performed as described in the accompanying examples.Sequencing of the described different RNA markers can be based on suitable cDNA libraries (DNA synthesized from an RNA population used as a template for sequencing) known in the art (e.g., Illumina TrueSeq cDNA libraries or ClontechSMART cDNA libraries, described, for example, at https: / / www.takarabio.com / products / cdna-synthesis / cdna-synthesis-kits / library-construction-kits). Sequencing can be generally performed by RNA sequencing methods known in the art and, inter alia, described, for example, in the Illumina TrueSeq cDNA library preparation protocol (https: / / support.illumina.com / content / dam / illumine-support / documents / documentation / chemistry_documentation / samplepreps_truseq / truseqrna / truseq-rna-sample-prep-v2-guide-15026495-f.pdf). Preferably, RNA sequencing is performed as described in the attached examples.

[0058] As mentioned above, in the context of multiplex sequencing of RNA, a cDNA library may be prepared. Therefore, it is preferred that 3' adapter molecules comprising barcodes are used for the preparation of the cDNA library. Preferably, these 3' adapter molecules comprising barcodes are preadenylated. It is also preferred that these 3' adapter molecules comprising barcodes are a collection. These 3' adapter molecules comprising barcodes, which may preferably be preadenylated, are either pooled or not, but are preferably pooled. More precisely, the collection of these 3' adapter molecules comprising barcodes, which may preferably be preadenylated, may preferably be pooled or not, but is preferably a pooled collection. Such a collection comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more types of 3' adapter molecules comprising barcodes and which may preferably be preadenylated.

[0059] In fact, to our great surprise, we have observed that when 3' adapter molecules are used for cDNA library preparation in the context of performing multiplex RNA sequencing, 3' adapter molecules containing barcodes reduce ligation and technical bias in RNA sequencing.In fact, in contrast to the present invention, the preparation of cDNA libraries for RNA sequencing in the prior art is carried out by using 3' adapter molecules that do not have barcodes.In the case of cDNA library preparation as carried out in the prior art, the barcodes are only ligated later during the preparation of the cDNA library.

[0060] As shown in FIG. 7, cDNA libraries made with standard 3' adapters (samples I-1 to I-3) when compared with cDNA libraries with 3' adapters containing barcodes, e.g. pooled MDX barcoded pre-adenylated 3' adapters) (MDX-4a to c) and non-pooled MDX barcoded pre-adenylated 3' adapters (MDX-7 to MDX-9), do not reduce ligation bias as shown by density estimates (shown as dotted lines at 0) close to the expected read counts, whereas 3' adapters used in the context of the method of the invention, which contain barcodes and may preferably be pre-adenylated, reduce ligation bias as shown by density estimates (shown as dotted lines at 0) close to the expected read counts. Thus, the 3' adapters of the invention are advantageous for the preparation of cDNA libraries when performing multiplex RNA sequencing. This advantage could not have been anticipated.

[0061] The present invention further relates to kits for carrying out the methods described and provided herein.

[0062] Preferably, the kit comprises a glass slide, preferably a positively charged glass slide, a frame slide, a frame slide with a PET membrane, or a glass slide with a membrane. It is also preferred that the kit further comprises formaldehyde and / or paraffin.

[0063] The kit may also preferably contain EDC or ETT and / or hematoxylin and / or eosin.

[0064] The kit may also contain containers that are preferably siliconized and nuclease-free.

[0065] In addition, the kit preferably further comprises a probe for fluorescent in situ hybridization of RNA and / or a 3' adapter molecule containing a barcode for preparing a cDNA library from RNA. These 3' adapter molecules containing a barcode that may be included by the kit are preferably preadenylated.

[0066] The RNA FISH probes preferably included by the kit may be preferably specific for HER2, ER, PR, NUPR1, CSF1, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, ER, PR, Bcl2, SCUBE2, stromelysin 3, cathepsin L2, GSTM1, BAG1, and CD68.

[0067] Moreover, the probes for RNA FISH, which are preferably further included in the kit, may preferably be specific for miR-21, miR-29a, miR-221, and miR-375.

[0068] Similarly, the probes for RNA FISH, which are preferably further included by the kit, may preferably be specific for U2 snRNA and / or 7SL scRNA.

[0069] Preferably, as controls, the kit may also contain probes specific for 28S rRNA, poly(A) RNA, β-actin, GAPDH, RPLPO, GUS, TFRC.

[0070] It is also preferred that the 3' adapter molecule comprising a barcode that may be included by the kit is a collection of 3' adapter molecules comprising a barcode. Preferably, the 3' adapter molecules comprising a barcode included by the kit are pooled or unpooled, but preferably are not pooled.

[0071] The present invention further relates to the use of such kits.

[0072] The aspects that characterize the invention are described herein, illustrated in the drawings, illustrated in the Examples, and reflected in the claims.

[0073] It should be noted that, as used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly indicates otherwise.Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that can modify or replace the method described herein.

[0074] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0075] The term "and / or" wherever used in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by this term".

[0076] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, more preferably within 5%, and most preferably within 3% of a given value or range.

[0077] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a referred integer value or step or group of integer values ​​or steps, but not the exclusion of any other integer value or step or group of integer values ​​or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including", or, as sometimes used herein, with the term "having".

[0078] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0079] Unless specifically indicated otherwise, in each instance herein, the terms "comprising," "consisting essentially of," and "consisting of" can all be replaced with either of the other two terms. For example, when a given feature, compound, or range is indicated as "comprised by" a respective broader term, such broader term can also "consist of" such feature, compound, or range.

[0080] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0081] All publications and patents cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference is inconsistent or contradictory with the present specification, the present specification will take precedence over any such material. [The present invention 1001] (a) determining the presence or absence of mRNA species and / or at least one miRNA species of disease-associated biomarkers in a sample obtained from a subject by multiplex fluorescent in situ hybridization (FISH); and (b) determining whether the mRNA species of the disease-associated biomarkers and / or the miRNA species of the disease-associated biomarkers of step (a) are present in the sample by multiplex sequencing. A method for diagnosing a disease comprising: The method, wherein that portion of the sample determined in step (a) to be present with the mRNA species and / or at least one miRNA species of a disease-associated biomarker is subjected to laser capturing prior to performing step (b). [The present invention 1002] 1001. The method of claim 1001, wherein step (a) further comprises determining whether snRNA and / or scRNA species of disease-related biomarkers are present in said sample by multiplex FISH. [The present invention 1003] Any of the methods of the invention, wherein step (b) further comprises determining whether snRNA and / or scRNA species of disease-associated biomarkers are present in the sample by multiplex sequencing. [The present invention 1004] Any of the methods of the invention, wherein the disease is indicated by the presence of disease-associated biomarkers in steps (a) and (b). [The present invention 1005] Any of the methods of the invention, wherein the presence or absence of said disease-associated biomarker allows for the diagnosis of disease. [The present invention 1006] Any of the methods of the preceding claims, wherein the disease is cancer. [The present invention 1007] The method of claim 1006, wherein the cancer is breast cancer. [The present invention 1008] Any of the methods of the present invention, wherein the mRNA is selected from the group consisting of HER2, ER, PR, NUPR1, CSF1, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, ER, PR, Bcl2, SCUBE2, stromelysin 3, cathepsin L2, GSTM1, BAG1, and CD68. [The present invention 1009] Any of the aforementioned methods of the present invention, wherein the miRNA is selected from the group consisting of miR-21, miR-29a, miR-221, and miR-375. [The present invention 1010] Any of the methods of the present invention, wherein the snRNA is U2 snRNA. [The present invention 1011] Any of the methods of the present invention, wherein the scRNA is 7SL scRNA. [The present invention 1012] Any of the methods of the invention, wherein one or more additional RNAs selected from the group consisting of 28S rRNA, poly(A) RNA, β-actin, GAPDH, RPLPO, GUS, TFRC are determined by FISH as a control. [The present invention 1013] Any of the methods of the present invention, wherein the laser capturing is laser capture microdissection. [The present invention 1014] Any of the methods of the preceding invention, wherein the sample is a tissue section sample. [The present invention 1015] Any of the methods of the invention, wherein the sample is formaldehyde fixed. [The present invention 1016] Any of the methods of the invention, wherein the sample is paraffin embedded. [The present invention 1017] Any of the methods of the invention, wherein the sample is obtained by microtome sectioning of a paraffin-embedded sample. [The present invention 1018] The method according to claim 1017, wherein said sample obtained by microtome sectioning is incubated at a temperature between 25°C and 0°C after obtaining. [The present invention 1019] Any of the methods of the invention, wherein the paraffin-embedded sample is deparaffinized prior to step (a). [The present invention 1020] Any of the methods of the invention, wherein the sample is fixed prior to step (a). [The present invention 1021] The method of claim 1020, wherein said sample is fixed by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 5-ethylthio-1H-tetrazole (ETT). [The present invention 1022] Any of the preceding methods of the invention, wherein when multiplex FISH is performed, fluorescent signals are detected by microscopy. [The present invention 1023] Any of the aforementioned methods of the invention, wherein probes attached by click chemistry are used for fluorescent in situ hybridization (FISH) of RNA. [The present invention 1024] Any of the methods of the invention, wherein the sample is stained with hematoxylin and eosin after FISH of RNA is performed. [The present invention 1025] Any of the methods of the invention, wherein RNA is extracted from the sample where multiplex sequencing of RNA is performed. [The present invention 1026] Any of the methods of the invention, wherein the sample is checked for the ratio of polyA mRNA and 28S rRNA prior to carrying out step (a) and / or step (b) of the method. [The present invention 1027] Any of the methods of the invention, wherein prior to carrying out step (a) and / or step (b) of the method, the sample is checked whether the ratio of polyA mRNA to 28S rRNA is 1:1 or greater than 1:1, preferably 1.05:1, 1.1:1, 1.15:1, 1.2:1. [The present invention 1028] Any of the preceding methods of the invention, wherein for multiplex sequencing of RNA, a cDNA library is prepared by using a 3' adapter molecule comprising a barcode. [The present invention 1029] The method of any one of claims 10 to 15, wherein the 3' adapter molecule comprising a barcode is preadenylated. [The present invention 1030] The method of any one of claims 1028 to 1029, wherein the 3' adapter molecule comprising a barcode is an aggregate. [The present invention 1031] The method of any of claims 1028 to 1030, wherein the 3' adapter molecules comprising barcodes are pooled. [The present invention 1032] A kit for carrying out any of the methods of the present invention. [The present invention 1033] The kit of the present invention 1032 comprising a slide, preferably a positively charged slide, a frame slide, a frame slide with a PET membrane, or a slide with a membrane. [The present invention 1034] The kit of any one of claims 1032 to 1033, further comprising formaldehyde and / or paraffin. [The present invention 1035] The kit of any of claims 1032 to 1034, comprising a probe for fluorescent in situ hybridization of RNA, and / or a 3' adapter molecule comprising a barcode for preparing a cDNA library from RNA. [The present invention 1036] The kit of the present invention, wherein the 3' adapter molecule comprising a barcode is preadenylated. [The present invention 1037] The kit of any one of claims 1035 to 1036, wherein the 3' adapter molecule comprising a barcode is an aggregate. [The present invention 1038] The kit of any of claims 1035 to 1037, wherein the 3' adapter molecule comprising a barcode is not pooled. [The present invention 1039] The kit according to any one of claims 1032 to 1038, comprising an EDC or an ETT. [The present invention 1040] The kit according to any one of claims 1032 to 1039, comprising hematoxylin and / or eosin. [The present invention 1041] The kit of any one of claims 1035 to 1040, wherein the probes for FISH of RNA are specific to HER2, ER, PR, NUPR1, CSF1, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, ER, PR, Bcl2, SCUBE2, stromelysin 3, cathepsin L2, GSTM1, BAG1, and CD68. [The present invention 1042] The kit of any one of claims 1035 to 1041, wherein the probe for RNA FISH is specific to miR-21, miR-29a, miR-221, and miR-375. [The present invention 1043] The kit of any of claims 1035 to 1042, wherein the probe for RNA FISH is specific to U2 snRNA. [The present invention 1044] The kit of any of claims 1035 to 1043, wherein the probe for RNA FISH is specific to 7SL scRNA. [The present invention 1045] The kit according to any one of claims 1035 to 1044, wherein the probe for FISH of RNA is specific for 28S rRNA, poly(A) RNA, β-actin, GAPDH, RPLPO, GUS, TFRC as a control. [The present invention 1046] A kit according to any one of claims 1032 to 1045, comprising a siliconized, nuclease-free container. [The present invention 1047] Use of any one of the kits according to the present inventions Nos. 1032 to 1046 for carrying out any one of the methods according to the present inventions Nos. 1001 to 1031. [Brief description of the drawings]

[0082] The figure shows: [Figure 1]Distinguishing FFPE breast cancer cell lines using RNA FISH and shorter DNA probes. Staining and visualization were performed in parallel using 45 DNA (24–30 nt), 39 DNA (14–21 nt) or 53 (11–15 nt) LNA / DNA probes conjugated to ATTO-550 to distinguish HER2-positive (HCC-1954) and HER2-negative (MDA-MB231) breast cancer cell lines. Five rRNA probes were conjugated to ATTO-488. Probes were hybridized at 40°C with 25% formamide and 1 M NaCl. The HCC-1954 cell line could not be distinguished from MDA-MB231 due to rRNA mishybridization of the DNA probe. However, the LNA / DNA probes conclusively discriminated between HER2+ and HER2- cells, indicating that these probes are specific. Exposure times for rRNA and HER2 were 80 ms and 200 ms, respectively. Scale bar, 50 μm. [Diagram 2] In situ click labeling. Exemplary setup. Two short fluorophore-labeled adjacent probes are tagged with azide and alkyne and fused by click chemistry. Mishybridized probes are removed after stringent washing. TM is measured for each approx. 15 nt long LNA / DNA probe (duplexed with RNA). Both TMs are similar and were at approx. 50°C in phosphate buffer containing 50% formamide / 1 M NaCl. [Diagram 3]RNA FISH and DAPI nuclear DNA staining of six ncRNAs, poly(A) RNA. RNA FISH was performed using the HCC-1954 cell line. We used 12-17 nt long LNA-modified DNA probes targeting U2 snRNA, 7SL small cytoplasmic (sc)RNA, U1 snRNA, mt-RNA, rRNA, U3 snoRNA, and a poly(T) probe targeting poly(A) conjugated to ATTO-488, ATTO-532, ATTO-Rho12, ATTO-Rho 14, ATTO-647N, ATTO-680, and ATTO-550, respectively. Cells were scanned using a Vectra Intelligent Slide Analysis System (PerkinElmer). [Figure 4] The MultiplexDX (MDX) method of sample processing compared to conventional (prior art) methods of sample processing. The method of the present invention inhibits diffusion of small RNAs (e.g., microRNAs) from cells, ensuring high concentrations of small RNAs. Mouse brain tissue sections (10 μm) were obtained using a microtome from either formalin-fixed, flash-frozen (FFFF) tissues that were processed immediately (0 s, baseline control) or formalin-fixed, paraffin-embedded (FFPE) tissues that were processed after 5, 60, 180, and 600 s. Only the MDX paraffin ribbons were briefly placed in an ice-cold water bath, then in a 42° C. water bath, and then placed on a glass slide after 5 s (MDX method, data points enclosed in shaded boxes) or after 60 s, 180 s, and 600 s (other methods). Note that the paraffin ribbons processed according to conventional (prior art) methods were not placed in an ice-cold water bath after they were obtained from microtome sectioning. Tissues were deparaffinized and dried, after which microRNA was extracted using the Ambion® PureLink® miRNA Isolation Kit and microRNA concentrations were measured using NanoDrop. [Figure 5A]Effect of RNA degradation. Effect of RNA degradation on polyA mRNA / 28S rRNA ratio after incubation of rat liver tissue for 0, 1, 3, 6, and 12 hours at either room temperature (25°C) or elevated temperature (37°C). The ratio of polyA mRNA / 28S rRNA (fluorescence activity / unit) can be used to assess RNA degradation in formalin-fixed paraffin-embedded (FFPE) tissues, with a ratio below 1 signifying degraded RNA (1.15 is marked as the dotted line as a presumably ideal ratio). [Figure 5B] Effect of RNA degradation. Effect of RNA degradation on the mean fluorescence intensity of polyA mRNA expression after incubation of rat liver tissue for 0, 1, 3, 6, and 12 hours at either room temperature (25°C) or elevated temperature (37°C). Confocal micrographs of polyA mRNA expression showing the reduction in fluorescence intensity across time points. [Figure 6] Laser capture microdissection (LMD) reduces bias introduced by sampling heterogeneous tissues and ensures concordance between RNA fluorescent in situ hybridization (FISH, A) and RNA sequencing (B). A, RNA FISH reveals Her2 amplification in breast cancer biopsy tissue in patient A (left panel) but not in patient B (right panel). B, Laser capture microdissection of the Her2+ cell population (~100 cells shown in the square inset) followed by RNA sequencing shows high concordance with Her2 expression assessed by RNA FISH. C, Whole tissue sampling followed by RNA sequencing demonstrates low concordance with RNA FISH due to sampling from a heterogeneous cell population. This may lead to an inaccurate diagnosis of Her2 status in patient A and, consequently, inappropriate or ineffective treatment. [Figure 7]Barcoded pre-adenylated 3'-adapters reduce ligation (A) and technical (B) bias in small RNA sequencing. A, Violin diagram illustrating density estimates of small RNA sequencing libraries prepared with miRXplore Universal Reference containing an equimolar pool of 962 unique microRNA sequences. Libraries were generated using standard Competitor-I 3' adapters (samples I-1 to I-3), pooled MDX barcoded pre-adenylated 3' adapters (MDX-4a to c) or unpooled MDX barcoded pre-adenylated 3' adapters (MDX-7 to MDX-9). MDX 3' adapters reduce ligation bias as shown by density estimates closer to expected read counts (shown as dotted line at 0) compared to Competitor-I 3' adapters. B, Box plots demonstrate that pooling MDX-barcoded pre-adenylated 3' adapters reduces the technical bias observed for miR-21, miR-29a, miR-221, and miR-375 when using unpooled MDX-barcoded pre-adenylated 3' adapters. Note that pooled MDX-barcoded pre-adenylated 3' adapters are shown on the left and unpooled MDX-barcoded pre-adenylated 3' adapters are shown on the right. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] The following sequences are provided herein: TIFF0007618234000003.tif38128TIFF0007618234000004.tif244128TIFF0007618234000005.tif124128TIFF0007618234000006.tif152128

[0084] The present invention is also characterized by the following items. 1. (a) determining the presence or absence of mRNA species and / or at least three miRNA species of disease-associated biomarkers in a sample obtained from a subject by multiplex fluorescent in situ hybridization (FISH); and (b) determining whether the mRNA species of the disease-associated biomarkers and / or the miRNA species of the disease-associated biomarkers of step (a) are present in the sample by multiplex sequencing. A method for diagnosing a disease comprising: 2. The method of item 1, wherein step (a) further comprises determining whether snRNA and / or scRNA species of disease-associated biomarkers are present in the sample by multiplex FISH. 3. The method of any of the preceding items, wherein step (b) further comprises determining whether snRNA and / or scRNA species of disease-associated biomarkers are present in the sample by multiplex sequencing. 4. The method of any of the preceding items, wherein the presence of disease-associated biomarkers in step (a) and step (b) is indicative of the disease. 5. The method of any of the preceding items, wherein the presence or absence of said disease-associated biomarker allows for the diagnosis of disease. 6. The method of any of the preceding items, wherein the disease is cancer. 7. The method of item 6, wherein the cancer is breast cancer. 8. The method of any of the preceding items, wherein the mRNA is selected from the group consisting of HER2, ER, PR, NUPR1, CSF1, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, ER, PR, Bcl2, SCUBE2, stromelysin 3, cathepsin L2, GSTM1, BAG1, and CD68. 9. The method of any of the preceding items, wherein the miRNA is selected from the group consisting of miR-21, miR-29a, miR-221, and miR-375. 10. The method of any of the preceding items, wherein the snRNA is U2 snRNA. 11. The method of any of the preceding items, wherein the scRNA is 7SL scRNA. 12. The method of any of the preceding items, wherein one or more additional RNAs selected from the group consisting of 28S rRNA, poly(A) RNA, β-actin, GAPDH, RPLPO, GUS, and TFRC are determined by FISH as a control. 13. A kit for carrying out the method of any of the preceding items. 14. Use of the kit according to item 13 for carrying out any one of the methods according to items 1 to 12.

[0085] The present invention is further described by the following examples, but is not limited to the examples or any particular aspects thereof. EXAMPLES

[0086] Probe Design Testing To test the RNA FISH probe design and compare it with other commercially available designs, two breast cancer cell lines, HER2+ (HCT1954) and HER2- (MDA-MB231), were used. The Stellaris probe design program (Biosearch Technologies) was used to prepare the ERBB2 mRNA probe. Instead of the 20 nt long probe, 45 24-30 nt long DNA probes were designed to increase the binding affinity and were then used to detect the T M The length of the probe was varied to equalize (the resulting T MThe melting temperatures varied from 48.1 to 56.9 °C in 50% formamide (FA), 1 M NaCl, 50 mM phosphate (pH 7.0). The GC content was limited to 50-64% as suggested by Stellaris. In the subsequent RNA FISH, rRNA probes were also used as internal controls and standards for signal specificity and RNA content. Then, DNA probes (24-30 nt long) were shortened from either the 5' or 3' end to avoid rRNA mishybridization (segments not longer than 8 nt with rRNA sequence complementarity), resulting in 39 short DNA probes (14-21 nt long). The melting temperatures of these DNA probes varied from 38.8 to 47.1 °C. Although a small discrimination was observed between HER2+ and HER2- breast cancer cells, rRNA mishybridization still predominated. To avoid mishybridization and increase the specificity of the probes, 53 short (11-15 nt long) directly labeled LNA-modified DNA (LNA / DNA) oligonucleotide probes were synthesized for ERBB2 mRNA with sequence segments within 6 nt that cross-hybridize with the most abundant RNAs (rRNA, tRNA, snRNA, mitochondrial rRNA, etc.) using the RNA FISH probe design described herein. The melting temperatures of these LNA / DNA probes varied from 44.2 to 52.1°C. These LNA / DNA probes were conclusively shown to be specific and differentiated between HCC-1954 (HER2+) and MDA-MB231 (HER2-) breast cancer cells (see Figure 1). Furthermore, when comparing the signal intensity of 100 cells from mRNA FISH and the copy number of ERRB2 transcripts from mRNA deep sequencing of the two HER2+ and HER2- cell lines, the difference from mRNA FISH was 51-fold compared to the difference from mRNA sequencing of 49-fold. This indicates that a very high degree of specificity was achieved.

[0087] RNA FISH: Note 1: The proteinase K permeabilization, 4% PFA fixation, acetylation and endogenous biotin blocking steps may be omitted. NOTE 2: If FFFF (formaldehyde fixed, fresh frozen) tissue is used, after thawing and air drying, incubate the slides in 50 ml of 1-methylimidazole buffer (pH 8.0) at 25°C for 2 min, start EDC fixation immediately, and proceed to step 14.

[0088] Microtome sectioning 1 Paraffin blocks were trimmed to an optimal cut surface containing the sample. 2. 5 μm slices were cut. A brush was used to draw the sections onto the knife holder. 3. The paraffin ribbon or section (slice) was placed in an ice-cold water bath with a second wet brush (so it would expand and wrinkles would disappear). 4 Using a glass slide, remove the floating paraffin sections, place the paraffin ribbon in a 42°C water bath until it expands and wrinkles disappear, then remove the paraffin section with a second wet brush and place the section. Note: Using a water bath with a temperature higher than 42°C may cause diffusion of RNA, especially short RNA, out of the tissue as the paraffin melts. 5 Sections were allowed to dry at room temperature for at least 1 h until the slide was completely dry and any water trapped between the tissue and the slide had evaporated. 6 The sections were baked at 56°C for 1 hour.

[0089] Deparaffinization 7 Slides were incubated twice in 50 ml of Histo-Clear II for 5 minutes at 25°C. 8 Slides were incubated in 50 ml of 100% ethanol at 25°C for 2 minutes. 9 Slides were incubated twice in 50 ml of 95% ethanol at 25°C for 1 minute. 10 Slides were incubated in 50 ml of 70% ethanol at 25°C for 1 minute. 11 The slides were incubated in 50 ml of 50% ethanol at 25°C for 1 minute. 12 Slides were rinsed by keeping them in a Coplin jar under cold running tap water.

[0090] Fixation of tissue with EDC / 5-ETT NOTE 3: When targeting long RNAs (mRNA and rRNA), EDC fixation can be omitted, but it is necessary to ensure that the tissues used are well preserved and do not contain high levels of hydrolyzed RNA. Note 4: EDC fixation may be advantageous when targeting tRNAs that contain a 5' phosphate. 13 The slides were incubated in 50 ml of 1-methylimidazole buffer (pH 8.0) for 2 min at 25° C. This step was to remove residual 1×TBS buffer. 14 Fresh EDC / 5-ETT solution was made before use. 15 In a humid chamber, the slides were placed face up in a slide rack. The 1-methylimidazole buffer (pH 8.0) was removed by tilting the slides and decanting the solution. In a humid chamber, the slides were placed face up in a slide rack. 500 μl of 16 EDC / 5-ETT solution was added to each slide and the samples were incubated at 50° C. for 3 hours in a closed humidified chamber.

[0091] Post-fixation wash steps 17 The samples were washed twice with 50 ml of 1× TBS for 3 minutes at 25°C.

[0092] Prehybridization and hybridization of probes and RNA 18 Slides were placed tissue side up in a slide rack in a humidified chamber. 19 500 μl of freshly prepared hybridization buffer was added to each slide within the hydrophobic barrier. The tissue was completely covered. Slides were incubated for 1 hour at 25° C. in a closed humidified chamber. 20 The hybridization buffer was removed by tilting the slide and decanting the solution.

[0093] Probe preparation and hybridization 21 Select the desired probe and set the oven to the appropriate hybridization temperature, T M The oven was preheated to a temperature approximately 20°C lower than that of the oven. 22 500 μl of hybridization solution containing the probes was added to each slide: concentrations of mRNA probe: 4 nM, abundant ncRNA: 10 nM, rRNA probe: 20 nM. 23 The slides were incubated in a closed humidified chamber at the hybridization temperature (37–50°C) for 6–16 h.

[0094] Post-hybridization washes 24 Samples were washed twice for 5-10 min at 25°C in a glass Coplin jar filled with 50 ml of Wash Buffer 1. Note: If hybridization buffer (50% FA) is used, use Wash Buffer 1 (50% FA), but if hybridization buffer (25% FA) is used, use Wash Buffer 1 (25% FA). 25 Samples were washed in 50 ml of wash buffer 2 at 25°C for 5 minutes. 26 Samples were rinsed in 50 ml of 1× TBS-T buffer at 25°C for 3 min.

[0095] In situ click chemistry cross-linking 27 In an Eppendorf tube (for one slide), a solution of 63 μl of 20 mM CuSO4 and 125 μl of 50 mM ligand THPTA was prepared. The final concentration of copper in the tube was 0.25 mM, and the final concentration of ligand 1 in the tube was 1.25 mM (ligand to copper ratio, 5:1). 28 250 μl of 100 mM sodium ascorbate was added. The final concentration of sodium ascorbate in the tube was 5 mM. 29 The tube was closed and mixed by inverting the tube several times (to prevent more oxygen from diffusing out). 30 400 μl of freshly prepared CuSO4 / THPTA / sodium ascorbate solution was added to each slide. 31 Slides were incubated for 1–2 h at room temperature in a closed humidified chamber, away from light. 32 The samples were washed twice with 50 ml of 1×TBS-T at 25° C. for 3 minutes.

[0096] Stringent washes, if required 32a 1 ml of wash buffer was added to each slide. Slides were incubated for 5-10 min at 40-50°C (or 0-10°C higher than the previous hybridization temperature) in a closed humidified chamber. Note: If hybridization buffer (50% FA) is used, use wash buffer 1 (50% FA), but if hybridization buffer (25% FA) is used, use wash buffer 1 (25% FA). 32b Samples were washed in 50 ml of wash buffer 2 at 25°C for 5 minutes. 32c Samples were rinsed in 50 ml of 1×TBS-T buffer at 25° C. for 3 min.

[0097] Mounting slides for microscopy 33 Slides were placed face up horizontally in a humidified slide rack and 500 μl of working DAPI solution was added to each slide for 10 minutes at 25°C. 34 Slides were washed twice in 50 ml of 1× TBS at 25° C. for 3 minutes. 35 The slide was placed face up horizontally in a humidified slide rack and two drops of mounting solution were added onto the tissue section. 36 A glass coverslip was carefully placed over the tissue section. 37 The cover glass slides were air dried for 10 minutes. The 38 coverglass slides were stored in a slide rack in the dark.

[0098] Fluorescent slide imaging 39 For use of the Perkin Elmer Vectra 3.0 Quantitative Pathology Imaging System, including slide scanning, RNA visualization, RNA biomarker quantification, spectral imaging, demixing, multiplex visualization, whole slide scanning, image analysis, and more, see the Instructions for Use: Please see https: / / www.perkinelmer.com / Content / LST_Software_Downloads / Vectra-User-Manual-3-0-3.pdf.

[0099] Laser Capture Microdissection: Tissue Microdissection and RNA Extraction 40 Five-μm tissue sections previously stained and visualized by RNA FISH were hematoxylin and eosin (H&E) stained for further pathological re-examination. 41 Tissue areas (regions) of interest were identified by microscopy and comparison of RNA FISH and H&E images, circled by the imaging software, and these H&E slides were subsequently laser microdissected by a Leica LMD7 (see https: / / www.leica-microsystems.com / applications / life-science / laser-microdissection / ). 42 Five to ten 5-μm sections were microdissected prior to RNA extraction in siliconized, nuclease-free Eppendorf tubes. 43 RNA extraction was performed on matched case‐control specimens on the same day as tissue microdissection to prevent tissue desiccation, RNA damage and technical variance. 44 500 μl of Trizol@reagent was added to the Eppendorf tube containing the tissue pieces, the tissue was homogenized by pipetting up and down, and the tissue was incubated at room temperature for 5 minutes. 45 200 μl of chloroform was added, vigorously shaken (vortexed) for 15 seconds, and the samples were incubated at room temperature for 3 minutes. 46 Samples were centrifuged at 11,500 rpm (12,568 × g) for 10 min at 4°C using a Sorvall Fresco centrifuge. After phase separation, the clear upper aqueous phase containing the RNA was collected without disturbing the cloudy interphase or the lower colored layer. The pH of the aqueous phase was set to acidic (approximately 4). 48 The aqueous phase was transferred and divided into two new tubes, each containing approximately 500 μl of lysate. 49 500 μl of ice-cold isopropyl alcohol was added to each of the tubes, incubated on ice for 30 min, and the samples were centrifuged at 11,500 rpm (12,568×g) for 10 min at 4°C. The supernatant was removed. The tube was then placed back into the centrifuge in the same orientation and spun for an additional 10 seconds to collect the remaining liquid. For small pellets, no washing of the pellet was necessary. 51 For larger pellets, 500 μl of ice-cold 75% EtOH solution was added, the tube was inverted once without disturbing the pellet, and centrifuged at 11,500 rpm (12,568 × g) for 10 min at 4 °C to collect the liquid, remove the liquid, and centrifuge again to remove residual liquid. 52 The supernatant was completely removed (i.e., with an additional 10 second spin). The RNA sample was redissolved in 100 μl of Millipore water and the volume was recombined. 53 RNA was quantified using a Bioanalyzer (Agilent, Danbury, CT, USA) total RNA chip.

[0100] RNA sequencing: 54 RNA sequencing of RNA extracted from microdissected tissues (1–2 μg) was performed using the Illumina TrueSeq cDNA library preparation protocol: https: / / support.illumina.com / content / dam / illumina-support / documents / documentation / chemistry_documentation / samplepreps_truseq / truseqrna / truseq-rna-sample-prep-v2-guide-15026495-f.pdf.

Claims

1. (a) determining the presence or absence of mRNA species and / or at least one miRNA species of disease-associated biomarkers in a sample obtained from a subject by multiplex fluorescent in situ hybridization (FISH); and (b) determining whether the mRNA species of the disease-associated biomarkers and / or the miRNA species of the disease-associated biomarkers of step (a) are present in the sample by multiplex sequencing.

1. A method for detecting a disease-associated biomarker, comprising: subjecting that portion of the sample in which the mRNA species and / or at least one miRNA species of a disease-associated biomarker is determined to be present in step (a) to laser capturing and isolation prior to performing step (b) on the isolated cells; the laser capturing is laser capture microdissection; Before carrying out step (a) and / or step (b), the sample is checked for a ratio of polyA mRNA to 28S rRNA greater than 1; The method.

2. 2. The method of claim 1, wherein step (a) further comprises determining whether snRNA and / or scRNA species of disease-associated biomarkers are present in the sample by multiplex FISH.

3. 3. The method of claim 1 or 2, wherein step (b) further comprises determining whether snRNA and / or scRNA species of disease-related biomarkers are present in the sample by multiplex sequencing.

4. The method of any one of claims 1 to 3, wherein the disease is cancer.

5. The method of any one of claims 1 to 4, wherein the mRNA is selected from the group consisting of HER2, ER, PR, NUPR1, CSF1, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, Bcl2, SCUBE2, stromelysin 3, cathepsin L2, GSTM1, BAG1, and CD68, and the miRNA is selected from the group consisting of miR-21, miR-29a, miR-221, and miR-375.

6. The method of any one of claims 1 to 5, wherein the sample is a tissue section sample.

7. The method of any one of claims 1 to 6, wherein the sample is formaldehyde fixed.

8. The method of any one of claims 1 to 7, wherein the sample is paraffin-embedded.

9. The method according to any one of claims 1 to 8, wherein the sample is obtained by microtome sectioning of a paraffin-embedded sample.

10. The method of any one of claims 1 to 9, wherein a cDNA library is prepared for multiplex sequencing of RNA by using a 3' adapter molecule comprising a barcode.

11. A kit for carrying out the method of any one of claims 1 to 10, comprising: (a) a glass slide; (b) formaldehyde and / or paraffin; (c) a probe for multiplex fluorescent in situ hybridization of RNA; (d) a 3' adapter molecule comprising a barcode for preparing a cDNA library from RNA; (e) a probe specific for 28S rRNA; and (f) a probe specific for polyA mRNA.

12. Use of the kit according to claim 11 for carrying out the method according to any one of claims 1 to 10.

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