Fluorescence in situ hybridization method based on achieving super-resolution imaging

Abstract

Disclosed is a fluorescence in situ hybridization method based on achieving super-resolution imaging. The method comprises the steps of an expansion treatment of a sample to be tested, and in situ hybridization of the sample to be tested using a probe directed at a target of interest. The expansion treatment of the sample to be tested comprises the following: a1) and / or a2): a1) performing an expansion treatment on the sample to be tested before in situ hybridization; and a2) performing secondary expansion on the sample after the in situ hybridization. The probe directed at a target of interest is combined with expansion microscopy technology. After cell expansion, the volume increases about 60-fold compared to the original volume, so that the resolution effect of super-resolution microscopy can be realized, thus creating a more accurate detection site. Meanwhile, the problem that a better fluorescence detection result cannot be obtained due to weak fluorescence signals of existing commercial probes is avoided.

Description

A fluorescence in situ hybridization method based on super-resolution imaging

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application (application number 202410254373.5) filed on March 6, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biological detection, and in particular to a fluorescence in situ hybridization method based on achieving super-resolution imaging. Background Art

[0004] Fluorescence in situ hybridization (FISH) is a technique that uses fluorescently labeled nucleic acid fragments as probes to complement the DNA or RNA to be tested in tissues, cells or chromosomes, combining to form specific nucleic acid hybrid molecules. The location of the nucleic acid to be tested in tissues, cells or chromosomes is then displayed through a fluorescence detection system.

[0005] If the target nucleic acid on the cell or tissue section to be tested is homologous and complementary to the nucleic acid probe used, a hybrid of the target nucleic acid and the nucleic acid probe can be formed after denaturation, annealing, and renaturation. A specific nucleotide in the nucleic acid probe is labeled with a reporter molecule, such as biotin or directly labeled with fluorescein. This experimental method allows for qualitative, semi-quantitative, or relative localization analysis of the nucleic acid under a microscope, utilizing an immunochemical reaction between the reporter molecule and a specific avidin labeled with fluorescein, or directly using a fluorescence detection system.

[0006] Currently, there are two main methods for producing RNA-FISH fluorescent probes. One is to entrust the company to design and synthesize, which is relatively expensive; the other is to add the promoter required for RNA transcription to the template through PCR, and then transcribe to obtain the probe. This method is cumbersome.

[0007] Expansion microscopy (ExM) is a new super-resolution imaging technique that can improve the final image resolution by physically magnifying the organism itself, and can expand the volume of cells and tissues by 8,000 times. The core of this technology is to use expandable hydrogels to evenly embed biological samples in them, and then perform mechanical homogenization. Next, the gel expands by folding linear expansion, thereby also expanding the organism. This process provides anchoring for key biological molecules in the cell (such as organelles, cytoskeleton, etc.).

[0008] Expansion microscopy has significant application value in improving the resolution of optical microscopes. Using conventional fluorescence microscopy to image the expanded sample can also achieve super-resolution imaging. For example, it can be used to examine cells on a single-molecule basis, making the final observations more reliable and accessible. The process begins by filling the entire biological sample with acrylate and polymerizing it to form a gel. As the gel absorbs water and expands, biomolecules that were previously adjacent are pulled apart, but their relative positions remain unchanged.

[0009] With the help of expansion microscopy, the resolution of the sample can be greatly improved, making it possible to achieve super-resolution imaging using ordinary microscopes.

[0010] Although super-resolution imaging can be achieved by imaging the expanded sample using a conventional fluorescence microscope, the brightness of the fluorescence signal may be diluted after the sample expands, and the fluorescence signal is weak, which is not conducive to detection.

[0011] Zhang Xu, Gao Juntao, Zhang Qiwei and others provided a method for preparing DNA probes in "Method for preparing probes for target nucleic acid targets". This method is based on Tn5 and is very suitable for analyzing chromatin interactions within a distance of 100Kb or less.

[0012] Invention Disclosure

[0013] The present application provides a fluorescence in situ hybridization method based on achieving super-resolution imaging.

[0014] The fluorescence in situ hybridization method based on achieving super-resolution imaging of the present application includes the steps of expanding the sample to be tested and performing in situ hybridization with the sample to be tested using a probe targeting a target.

[0015] Wherein, the expansion treatment of the sample to be tested includes the following a1) and / or a2):

[0016] a1) Before in situ hybridization, the sample to be tested is subjected to expansion treatment;

[0017] a2) After in situ hybridization, the samples were subjected to a secondary expansion.

[0018] The expansion treatment method a1) is as follows: anchoring and polymerizing the sample to be tested, and then placing it in water until it is fully expanded. The above-mentioned full expansion means that the volume of the sample no longer increases.

[0019] The anchoring and polymerization method comprises placing the fixed sample in a paraformaldehyde / acrylamide buffer, allowing it to stand, removing the sample, placing it in a mixed solution containing a monomer solution, tetramethylethylenediamine, and acrylamide, and incubating it; the volume ratio of the monomer solution, tetramethylethylenediamine, and acrylamide in the mixed solution is 90:5:5; the monomer solution is a 1×PBS buffer containing 23% (w / v) sodium acrylate, 10% (w / v) acrylamide, and 0.1% (w / v) N,N′-methylenebisacrylamide; and the paraformaldehyde / acrylamide buffer is a 1×PBS buffer containing 0.7% paraformaldehyde and 1% acrylamide.

[0020] The expansion treatment method a2) is as follows: adding water to the sample after in situ hybridization, allowing the sample to swell a second time, and discarding the water after the expansion is complete.

[0021] Wherein, the preparation method of the probe for the target is:

[0022] b1) obtaining a target cDNA sequence corresponding to the target RNA;

[0023] b2) using a transposase to fragment the target cDNA sequence and adding adapter sequences to both ends of the fragmented DNA sequence;

[0024] b3) using the linker sequence to obtain the fragmented DNA sequence to generate a probe.

[0025] The above-mentioned method for preparing probes for the target is independent or minimally dependent on the specificity of the initial RNA sequence and can effectively remove regions of unwanted sequences (especially repetitive sequences), thereby making the method independent of species-specific Cot-1 DNA for blocking repetitive fragments.

[0026] An important advantage is that the amount of cDNA template required for probe preparation in this method is approximately 50 ng (e.g., 30 ng, 35 ng, 40 ng, 45 ng, 55 ng, 60 ng), which is much lower than the 1 μg required for traditional FISH. At the same time, for each site, only one fragmentation with the Tn5 high-efficiency transposase is required to prepare a large number of probes. The process is simple, efficient, and cost-effective.

[0027] In the present application, the target cDNA sequence can be derived from any sample containing the target cDNA.

[0028] The term "sample" is used in its broadest sense. In a kind of meaning, it means including cells (for example, people, bacteria, yeast and fungi), tissues or living bodies or samples or cultures and biological samples obtained from any source. Biological samples can be obtained from animals (including people) and refer to biological materials or compositions found therein, including but not limited to bone marrow, blood, serum, platelets, plasma, interstitial fluid, urine, cerebrospinal fluid, nucleic acid, RNA, tissue and its purification or filtered form. However, these examples should not be construed as limiting the sample types that can be used for the application.

[0029] In some embodiments, the sample is whole genome RNA.

[0030] In some embodiments, the transposase is hyperactive.

[0031] As used herein, the term "nucleic acid" refers to any molecule comprising nucleic acid, including but not limited to DNA or RNA. The term encompasses sequences comprising any known base analogs of DNA and RNA, including but not limited to: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl Q nucleoside, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, Q nucleoside, 2-thiocytosine and 2,6-diaminopurine.

[0032] In some embodiments, the target cDNA sequence of the target of interest is obtained by excluding unwanted sequence regions from the initial sequence.

[0033] As used herein, the term "region of unwanted sequences" refers to a region that is substantially free (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% free) of unwanted nucleic acids. Unwanted nucleic acids include, but are not limited to, repetitive nucleic acids, non-conservative sequences, conserved sequences, GC-rich sequences, AT-rich sequences, secondary structures, non-coding sequences (e.g., promoters, enhancers, etc.), or coding sequences.

[0034] In some embodiments, the unwanted region is selected from repetitive sequences.

[0035] In some embodiments, the method of eliminating is to amplify the target cDNA sequence of the target;

[0036] In some embodiments, the amplification is PCR amplification.

[0037] In some embodiments, the region of unwanted sequence is at least 100bp; or 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1500bp, 2000bp, 3000bp, 4000bp, 5000bp, 6000bp, 7000bp, 8000bp, 9000bp, 10000bp, 20000bp, 30000bp, 40000bp, or 50000bp.

[0038] In some embodiments, the transposase is selected from one or a combination of any two or more of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn9, Tn10, Tn551, Tn971, Tn916, Tn1545, Tn1681, Tgf2, Tol2, Himar1, and HARBI1.

[0039] Tgf2 and Tol2 are from the hAT family, Himar1 is from the Tcl / Mariner family, and HARBI1 is from the PIF / Harbinger family.

[0040] In some embodiments, the probe is labeled.

[0041] As used herein, the term "label" refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid or protein. Labels include, but are not limited to, dyes; radioactive labels such as 32P; a binding moiety such as biotin; a hapten such as digoxigenin; a luminescent, phosphorescent or fluorescent moiety; and a fluorescent dye alone or in combination with a moiety that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, etc. The label can be a charged moiety (positive or negative) or, alternatively, can be charge neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence comprising the label is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a label.

[0042] In some embodiments, the label is a fluorophore, a colorimetric label, a quantum dot, biotin, and other label molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cyclic olefins for click reactions, and initiator groups for polymer labeling), and can also be selected from polypeptides / protein molecules, LNA / PNA, non-natural amino acids and their analogs (such as peptoids), non-natural nucleic acids and their analogs (nucleotide mimics) and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced emission molecules, rare earth ion ligand molecules, polymetallic oxygen clusters, etc.).

[0043] In some embodiments, the label is a fluorophore.

[0044] In some embodiments, the fluorophore can be selected from fluorescein-based dyes, rhodamine-based dyes, and cyanine dyes.

[0045] In some embodiments, the fluorescein dye includes standard fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc.

[0046] In some embodiments, the rhodamine dyes include R101, tetraethyl rhodamine (RB200), and carboxytetramethylrhodamine (TAMRA).

[0047] In some embodiments, the cyanine dye is mainly selected from two categories, one is thiazole orange (TO), oxazole orange (YO) series and dimer dyes thereof, and the other is polymethine series cyanine dyes.

[0048] In some embodiments, the fluorophore may also be selected from the following dyes: diphenylethylene, naphthalimide, coumarins, acridines, pyrenes, and the like.

[0049] Fluorophores are typically labeled at the 5' end of the primer or probe sequence, but can also be placed at the 3' end by changing the modification bond (e.g., -OH or -NH bond).

[0050] In some embodiments, in step c), the method of generating the probe comprises amplification, cloning, synthesis, or a combination thereof.

[0051] The term "amplification" refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide when the term "nucleic acid" is used together, typically starting from a small amount of polynucleotide (e.g., as little as a single polynucleotide molecule), wherein the amplified product or amplicon is typically detectable. The amplification of polynucleotides includes a variety of chemical and enzymatic methods. Producing multiple DNA copies from one or several copies of a target cDNA or template DNA molecule during polymerase chain reaction (PCR), rolling circle amplification (RCA), or ligase chain reaction (LCR) is a form of amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, using reverse transcription RT-PCR to produce multiple cDNA molecules from a limited amount of RNA in a sample is a form of amplification. In addition, producing multiple RNA molecules from a single DNA molecule during the transcription process is also a form of amplification.

[0052] In some embodiments, in step c), the method for generating the probe is: amplifying the fragmented DNA sequence using primers that can bind to the linker sequence.

[0053] The term "primer" refers to an oligonucleotide, whether naturally occurring in a purified restriction digest or synthetically produced, which, when placed under conditions that induce the synthesis of primer extension products complementary to a nucleic acid chain (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH), can serve as a starting point for synthesis. The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its chains before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer should be long enough to initiate the synthesis of extension products in the presence of an inducing agent. The precise length of the primer will depend on many factors, including the use of temperature, primer source, and method. For example, in some embodiments, primers range from 10-100 or more nucleotides (e.g., 10-300, 15-250, 15-200, 15-150, 15-100, 15-90, 20-80, 20-70, 20-60, 20-50 nucleotides, etc.).

[0054] In some embodiments, primers comprise additional sequences that do not hybridize to target nucleic acids.Term " primer " includes chemically modified primers, fluorescently modified primers, functional primers (fusion primers), sequence-specific primers, random primers, primers with specific and random sequences, and DNA and RNA primers.

[0055] In some embodiments, the primer is labeled.

[0056] In some embodiments, the label is defined as the term "label" above;

[0057] In some embodiments, the label is selected from a fluorophore, a colorimetric label, a quantum dot, or biotin; preferably a fluorophore.

[0058] As used herein, the term "hybridization" is used to refer to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of binding between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the Tm of the hybrid formed, and the G:C ratio within the nucleic acids. A single molecule containing a pair of complementary nucleic acids within its structure will be "self-hybridizing."

[0059] In some embodiments, the hybridization assay is in situ hybridization;

[0060] Preferably, the in situ hybridization is to perform 3D FISH labeling on the probe and the expanded target cells.

[0061] The present application also provides a method for RNA signal localization, using the above-mentioned fluorescence in situ hybridization method based on achieving super-resolution imaging.

[0062] The present invention also provides a method for detecting diseases caused by abnormal RNA expression, including the above-mentioned RNA signal localization method.

[0063] Among them, the diseases caused by abnormal RNA expression include: diseases caused by abnormal MYC mRNA expression, diseases caused by abnormal TP53 mRNA expression, diseases caused by abnormal EGFR mRNA expression, diseases caused by abnormal BRAF mRNA expression, diseases caused by abnormal HER2 mRNA expression and / or diseases caused by abnormal SRC mRNA expression.

[0064] Among them, the diseases with abnormal MYC mRNA expression include lymphoma, lung cancer, breast cancer, gastric cancer, colorectal cancer, cervical cancer, leukemia, neuroblastoma, retinoblastoma, and sarcoma;

[0065] Diseases with abnormal TP53 mRNA expression include breast cancer, lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer, hematological malignancies, and gliomas;

[0066] Diseases with abnormal EGFR mRNA expression include non-small cell lung cancer, head and neck cancer, colorectal cancer, brain cancer, and pancreatic cancer;

[0067] Diseases with abnormal BRAF mRNA expression include malignant melanoma, thyroid cancer, colorectal cancer, non-small cell lung cancer, pancreatic cancer, test tube cancer, esophageal cancer, breast cancer, and ovarian cancer;

[0068] Diseases with abnormal HER2 mRNA expression include breast cancer and gastric cancer;

[0069] Diseases with abnormal SRC mRNA expression include breast cancer, colorectal cancer, lung cancer, prostate cancer, gastric cancer, osteosarcoma, soft tissue sarcoma, renal cancer, familial macrognathia, and cardiovascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG1 is a schematic diagram of a process flow of an embodiment of the present application.

[0071] FIG2 shows the mRNA signals of POLR2A detected in the nucleus and cytoplasm using the Tn5-RNA-FISH method and the commercial probe in Example 1.

[0072] Figure 3 shows the lncRNA signal of MALAT1 RNA detected in the cell nucleus using the Tn5-RNA-FISH method and using a commercial probe;

[0073] FIG4 shows the detection of intracellular POLR2A and MALAT1 RNA signals using Tn5-RNA-FISH probe combined with expansion microscopy.

[0074] Best Mode for Carrying Out the Invention

[0075] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0076] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0077] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0078] The instruments involved in the following embodiments mainly include: a PCR instrument (Biored), a hybridization instrument (Abbott Thermobite), and a water bath.

[0079] Reagents in the following examples: DMEM medium (purchased from GIBCO), streptomycin / penicillin double antibody (purchased from GIBCO), trypsin (purchased from GIBCO), FBS (purchased from GIBCO), RNA extraction kit (purchased from Life Technology), reverse transcription kit (purchased from Thermo Scientific), Qubit RNA high sensitivity kit (purchased from Life Technology), Qubit DNA high sensitivity kit (purchased from Life Technology), AntiFade mounting medium (containing DAPI, purchased from Life Technology), Fixogum (purchased from Marubu), Tn5 transposase kit (purchased from Vazyme), HS-Taq (purchased from Takara), PCR product purification kit (purchased from Zymo), 37% hydrochloric acid (purchased from Sinopharm), Tris-HCl (purchased from Sigma), Triton-X100 (purchased from Sigma), ethanol (purchased from Sigma), dextran sulfate (purchased from Sigma), glycogen (purchased from Life Technology), commercial MALAT1 RNA-FISH probe (purchased from Stellaris), commercial POLR2A RNA-FISH probe (purchased from Stellaris), 20× SSC (purchased from Life Technology), deionized formamide (purchased from Solarbio), PBS (purchased from Solarbio), 3 M sodium acetate (purchased from Solarbio), 4% paraformaldehyde (purchased from Solarbio), NP-40 (purchased from Solarbio), AcX (Thermo-Fisher), Lablel-IT Amine Modifying Reagent (Mirus Bio, LLC), amide (sigma), N,N′-methylenebisacrylamide (sigma), sodium acrylate (sigma), tetramethylethylenediamine (sigma), ammonium persulfate (sigma), ammonium persulfate (sigma).

[0080] A549 cells (Wuhan Punuosai Biotechnology Co., Ltd.), POLR2A mRNA (NCBI Gene ID: 5430), and MALAT1 RNA (NCBI Gene ID: 378938) in the following examples.

[0081] Consumables in the following examples: SuperFrost slides (purchased from ThermoFisher), ThermoFisher 1.5# cover slips (purchased from ThermoFisher).

[0082] The method for preparing a probe for target RNA in this application comprises the following steps:

[0083] 1) Primer Design: Design amplification primers for the target RNA and send them to a synthesis company for synthesis. Fluorescently labeled primers are synthesized according to the sequence provided in the Tn5 kit.

[0084] 2) RNA extraction: For each cell type, 1×10 6 The cells were extracted according to the experimental steps of the RNA extraction kit, and the extracted RNA was quantified using Qubit.

[0085] 3) cDNA Preparation: Add 1 μg of total RNA, reverse transcription primer, dNTPs, 5× reverse transcription buffer, RNase inhibitor, and reverse transcriptase to a 50 μl PCR tube in a 20 μl reaction volume. Reverse transcription conditions: 25°C for 10 minutes, then 50°C for 30 minutes.

[0086] 4) Probe Template cDNA Amplification: Take 2 μl of the reverse transcription product from Step 3 as the template and add the diluted primers (amplification primers from Step 1) to a 50 μl PCR tube for PCR. PCR conditions were: 98°C for 3 min, followed by 30 cycles of (98°C for 30 s, 55°C for 30 s, 72°C for 3 min), 72°C for 5 min, and a 4°C hold. PCR products were purified using a recovery kit, quantified using a Qubit assay, and stored at -20°C.

[0087] 5) Tn5 Fragmentation: Take 50 ng of the DNA product from step 4 and add Tn5 enzyme and reaction buffer to a total volume of 50 μl. Heat in a 55°C water bath for 10 minutes. Purify the DNA using a PCR product recovery kit.

[0088] 6) PCR amplification and fluorescent labeling: All products from step 5 were subjected to PCR amplification. The PCR conditions were 75°C for 5 min, (98°C for 30 s, 55°C for 30 s, 72°C for 30 s) × 30 cycles, 72°C for 5 min, and maintained at 4°C. The DNA product was purified using a PCR product purification kit, and then 50 ng was taken as a template for PCR amplification and labeling using the fluorescently labeled primers in step 1). The PCR conditions were 98°C for 3 min, (98°C for 30 s, 55°C for 30 s, 72°C for 30 s) × 30 cycles, 72°C for 5 min, and maintained at 4°C. After the labeled product was quantified using Qubit, 2 μl of glycogen was added for 2 hours of -80°C ethanol precipitation (0.1 times the volume of 3M sodium acetate, 2.5 times the volume of anhydrous ethanol). The probe after alcohol precipitation was washed three times with 75% ethanol, the ethanol was evaporated, and the probe was resuspended in hybridization solution (2×SSC, 10% dextran sulfate, 50% deionized formamide) and stored at -20°C.

[0089] 7) Probe denaturation: Before use, take the probe out of the refrigerator, place it in a PCR instrument, heat it at 95°C for 5 minutes, and then immediately place it on ice for later use.

[0090] The method for using the above-mentioned probe to perform FISH detection of the RNA to be tested in cells based on expansion microscopy technology in this application comprises the following steps:

[0091] 1) Sample fixation: Fix the cells with 4% paraformaldehyde at room temperature for 10 minutes, wash twice with 1×PBS, then add 1 ml of 70% alcohol and place at 4°C for 10 minutes to permeabilize. After the end, discard the alcohol and add washing buffer (2×SSC, 10% deionized formamide) and let it stand at room temperature for 2-5 minutes. (Usually, 8*10 cells are fixed.) 4 Cells were dropped onto glass slides and fixed after one day of incubation.

[0092] 2) Anchoring and Polymerization: The fixed sample was placed in 1 mL of paraformaldehyde / acrylamide buffer (0.7% paraformaldehyde, 1% acrylamide in 1× PBS) and incubated at 37°C for 5 hours. After incubation, 90 μL of pre-chilled monomer solution (23% (w / v) sodium acrylate, 10% (w / v) acrylamide, and 0.1% (w / v) N,N′-methylenebisacrylamide in 1× PBS) was removed from a -20°C freezer. 5 μL of tetramethylethylenediamine and 5 μL of acrylamide were added, mixed rapidly by pipetting, and then added dropwise to a -20°C pre-chilled incubation chamber. The sample was removed, any remaining liquid aspirated, and placed at the bottom of the incubation chamber, with the cell surface in contact with the liquid. The chamber was placed on ice for 5 minutes and then incubated at 37°C for 1 hour.

[0093] 2) Sample expansion: After the incubation is complete, remove the coverslip and gel with tweezers and add them to the digestion buffer (NEB buffer 2 containing 1mg / ml proteinase K) at 37°C for 30 minutes. Then remove the digestion solution and place the coverslip with the gel in 50ml of water. Change the water every 30 minutes until the sample is fully expanded (about 3-4 times). Select a smooth surface on the gel and cut off about 1cm 2 The gel blocks were used for subsequent hybridization.

[0094] 3) In situ hybridization: Add 100 μL of probe solution (2 ng / μL) to the bottom of a confocal microplate. Place the gel in the hybridization solution, ensuring that the cell surface is in contact with the probe. Place the confocal microplate in a hybridization apparatus and incubate at 37°C overnight. The next day, discard the hybridization solution and add 1 mL of wash buffer. Incubate at 37°C in the dark for 30 minutes. After incubation, discard the solution and add 1 mL of DAPI stain (10 mg / mL). Incubate at 37°C in the dark for 30 minutes. Discard the DAPI stain. Add 1 mL of wash buffer and let stand at room temperature for 2-5 minutes. Discard the buffer and carefully remove any excess liquid with absorbent paper.

[0095] 4) Secondary Expansion: Add 2 ml of water to the confocal dish, shake gently, discard the water, and repeat the wash cycle. Then, add 2 ml of water to the dish and let it sit at room temperature for 30 minutes to allow the sample to swell a second time. After expansion, discard the water and carefully remove any excess liquid with absorbent paper. Store the sample at 4°C in the dark or photograph it directly.

[0096] 5) Fluorescence Imaging and Processing: Mounted slides were imaged using a fluorescence microscope, confocal microscope, or super-resolution microscope. This application used a Zeiss confocal microscope (model: LSM780) equipped with 405, 488, 568, 594, and 647 lasers and corresponding filter combinations, and a 63× ApoPLAN ​​NA1.4 oil immersion lens. The immersion oil used was Zeiss Immersion Oil F518, with a refractive index of 1.515 at 25°C. Images were acquired using ZEN SP2.3 and processed using FIJI (ImageJ core version: 1.52h).

[0097] The method for performing FISH detection (not based on expansion microscopy) of the RNA to be tested in cells using the above-mentioned probe or commercial probe in this application comprises the following steps:

[0098] 1. Cell fixation: Fix cells with 4% paraformaldehyde at room temperature for 10 minutes, wash with 0.1M Tris-HCl for 10 minutes, then permeabilize the membrane and digest RNA with 0.5% Triton-X100 containing 10μg / mL RNase A, incubate in a water bath at 37°C for 30 minutes, wash three times with PBS, and incubate with 0.1M hydrochloric acid solution at room temperature for 30 minutes; wash three times with PBS, then incubate in 50% deionized formamide 2×SSC solution at room temperature for 30 minutes, dehydrate with graded ethanol, and air-dry.

[0099] 2. In situ Hybridization: Mix 10 μL of probe solution (2 ng / μL) with cells, seal slides with Fixogum, and hybridize in a hybridizer (75°C for 5 min, 37°C overnight). The next day, wash cells three times with 0.3% NP-40 in 2×SSC at room temperature for 5 min each time. Then, mount the slides with AntiFade mounting medium and seal the solution around the edges of the slides with Fixogum. Store in the dark at 4°C or photograph directly.

[0100] 3. Fluorescence Imaging and Processing: Seal the slides and image them using a fluorescence microscope or confocal microscope. This application used a Zeiss confocal microscope (model: LSM780) equipped with 405, 488, 568, 594, and 647 lasers and corresponding filter combinations, and a 63× ApoPLAN ​​NA1.4 oil immersion lens. The immersion oil used was Zeiss Immersion Oil F518, with a refractive index of 1.515 at 25°C. The images were acquired using ZEN SP2.3 and processed using FIJI (ImageJ core version: 1.52h).

[0101] As shown in Figure 1, the technical route of one embodiment of the present application can be briefly summarized as follows: 1) transcribe total RNA into cDNA; 2) amplify the target cDNA using amplification primers; 3) fragment the target cDNA using transposase Tn5 to obtain target DNA fragments; 4) amplify the target DNA fragments using fluorescently labeled primers to obtain fluorescent probes; 5) expand the sample (cell) to be tested; 6) perform in situ hybridization between the fluorescent probe and the expanded sample; 7) perform fluorescence detection on the hybridized sample. (The sample can also be expanded a second time before fluorescence detection.)

[0102] Example 1

[0103] First, probes prepared using the Tn5 method were used to detect RNA localization in unexpanded samples to verify the accuracy and sensitivity of the probes prepared using the Tn5 method. The Tn5-RNA-FISH probe was then compared with a commercial RNA-FISH probe (kit purchased from Stellaris) in A549 cells targeting POLR2A mRNA to verify the site-specific labeling of Tn5-RNA-FISH.

[0104] Previous studies have shown that POLR2A mRNA is distributed in both the nucleus and cytoplasm. The results obtained using the Tn5-RNA-FISH method are the same as those obtained using commercial probes. POLR2A mRNA signals can be detected in both the nucleus and cytoplasm. The experimental results are shown in Figure 2.

[0105] Example 2

[0106] Probes prepared using the Tn5 method were used to detect RNA localized in the nucleus in unexpanded samples. Compared to commercial probes, Tn5-RNA-FISH probes are longer. Proper nuclear entry of the probes can be a potential challenge in this application. To test the nuclear entry ability of Tn5-FISH probes, a Tn5-FISH probe targeting MALAT1 RNA was used for validation. MALAT1 RNA is a lncRNA primarily localized in the nuclear speckle region of the cell nucleus.

[0107] The experimental results are shown in Figure 3. In A549 cells, the RNA signal of MALAT1 detected by Tn5-RNA-FISH was mainly located in the cell nucleus, and the signal localization was consistent with the signal localization detected by previous studies and commercial probes.

[0108] Example 3

[0109] Expansion microscopy has important applications in improving the resolution of optical microscopes. Using conventional fluorescence microscopy to image expanded samples can also achieve super-resolution imaging. However, sample expansion can dilute the brightness of the fluorescence signal, which places high demands on probe signal intensity and probe-target binding, requirements that cannot be met using existing commercial probes. The Tn5-RNA-FISH probe, combined with expansion microscopy, was used to detect intracellular POLR2A and MALAT1 RNA.

[0110] The experimental results are shown in Figure 4. The signal localization of POLR2A and MALAT1 RNA detected by Tn5-RNA-FISH in expanded A549 cells was consistent with that before expansion. POLR2A RNA was distributed in both the nucleus and cytoplasm, while MALAT1 RNA was primarily distributed in the nucleus.

[0111] This invention can be used to test clinical samples. Probes are prepared for target RNA, detecting the target RNA while simultaneously expanding the sample. This effectively increases the accessibility of the target molecule and the strength of the detection signal without disrupting the RNA structure. This process also makes RNA information, which is previously difficult to distinguish due to its close proximity, easier to analyze, resulting in more intuitive and clear test results.

[0112] The above-mentioned method of performing FISH detection on the RNA to be tested in cells can be used to detect samples of various diseases caused by abnormal RNA expression, including but not limited to the following diseases:

[0113] Detection of disease samples with abnormal MYC mRNA expression, such as lymphoma, lung cancer, breast cancer, gastric cancer, colorectal cancer, cervical cancer, leukemia, neuroblastoma, retinoblastoma, sarcoma, etc.

[0114] Detection of disease samples with abnormal TP53 mRNA expression, such as breast cancer, lung cancer (especially non-small cell lung cancer), pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer, hematological malignancies (acute myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome), glioma, etc.

[0115] Detection of disease samples with abnormal EGFR mRNA expression, such as non-small cell lung cancer, head and neck cancer, colorectal cancer, brain cancer, pancreatic cancer, etc.

[0116] Detection of disease samples with abnormal BRAF mRNA expression, such as malignant melanoma, thyroid cancer, colorectal cancer, non-small cell lung cancer, pancreatic cancer, test tube cancer, esophageal cancer, breast cancer, ovarian cancer, etc.

[0117] Detection of disease samples with abnormal HER2 mRNA expression, such as breast cancer, gastric cancer, etc.

[0118] Detection of disease samples with abnormal SRC mRNA expression, such as breast cancer, colorectal cancer, lung cancer, prostate cancer, gastric cancer, osteosarcoma, soft tissue sarcoma, renal cancer, familial macrognathia, cardiovascular disease, etc.

[0119] and many other diseases caused by abnormal RNA expression.

[0120] Industrial Applications

[0121] The probe preparation method used in this application is simple and fast, does not require expensive instruments and equipment, and can obtain highly accurate fluorescent probes for RNA-FISH through simple steps; the probe production cost is low, and the cost is reduced by 5-10 times compared to commercial probes or traditional methods of preparing probes. By combining the probe of this application with expansion microscopy technology, the volume of the cells expands to about 60 times the original size after expansion, which can achieve the resolution effect of super-resolution microscopy and more accurate detection sites. At the same time, it avoids the problem of weak fluorescence signals of the original commercial probes and the inability to obtain good fluorescence detection results.

Claims

1. A fluorescence in situ hybridization method based on super-resolution imaging, characterized in that: The method comprises the steps of swelling the sample to be tested and performing in situ hybridization with the sample to be tested using a probe targeting a target.

2. The fluorescence in situ hybridization method based on super-resolution imaging according to claim 1, characterized in that: The expansion treatment of the sample to be tested includes the following a1) and / or a2): a1) Before in situ hybridization, the sample to be tested is subjected to expansion treatment; a2) After in situ hybridization, the samples were subjected to a secondary expansion.

3. The fluorescence in situ hybridization method based on super-resolution imaging according to claim 2, characterized in that: The expansion treatment method a1) is: anchoring and polymerizing the sample to be tested, and then placing it in water until it is fully expanded.

4. The fluorescence in situ hybridization method based on super-resolution imaging according to claim 3, characterized in that: The anchoring and polymerization method comprises the following steps: placing the fixed sample in a paraformaldehyde / acrylamide buffer solution, allowing the sample to stand, taking out the sample, placing the sample in a mixed solution containing a monomer solution, tetramethylethylenediamine, and acrylamide, and incubating the mixture; the volume ratio of the monomer solution, tetramethylethylenediamine, and acrylamide in the mixed solution is 90:5:5; the monomer solution is a 1×PBS buffer solution containing 23% (w / v) sodium acrylate, 10% (w / v) acrylamide, and 0.1% (w / v) N,N′-methylenebisacrylamide; and the paraformaldehyde / acrylamide buffer solution is a 1×PBS solution containing 0.7% paraformaldehyde and 1% acrylamide.

5. The fluorescence in situ hybridization method based on achieving super-resolution imaging according to claim 2, characterized in that: The expansion treatment method a2) is as follows: adding water to the sample after in situ hybridization, allowing the sample to swell a second time, and discarding the water after the swelling is complete.

6. The fluorescence in situ hybridization method based on super-resolution imaging according to claim 1, characterized in that: The preparation method of the probe for the target is: b1) obtaining a target cDNA sequence corresponding to the target RNA; b2) using a transposase to fragment the target cDNA sequence and adding adapter sequences to both ends of the fragmented DNA sequence; b3) using the linker sequence to obtain the fragmented DNA sequence to generate a probe.

7. The method according to claim 6, characterized in that The transposase is selected from one or a combination of any multiple of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn9, Tn10, Tn551, Tn971, Tn916, Tn1545, Tn1681, Tgf2, Tol2, Himar1 and HARBI1.

8. The method according to claim 6, characterized in that The probe is labeled, and the label is selected from fluorophores, colorimetric labels, quantum dots, biotin, alkyne groups for Raman diffraction imaging, cyclic olefins for click reactions, initiator groups for polymer labeling, polypeptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs and non-natural nucleic acids and their analogs and nanostructures.

9. The method according to claim 6, characterized in that The primer is labeled, and the label is selected from fluorophores, colorimetric labels, quantum dots, biotin, alkyne groups for Raman diffraction imaging, cyclic olefins for click reactions, initiator groups for polymer labeling, polypeptide / protein molecules, LNA / PNA, non-natural amino acids and analogs thereof, non-natural nucleic acids and analogs thereof, and nanostructures.

10. The method according to claim 6, characterized in that In step b3), the method for producing the probe includes amplification, cloning, synthesis or a combination thereof; the amplification method is to amplify the fragmented DNA sequence using primers that can bind to the linker sequence.

11. A method for RNA signal localization, characterized in that: Use the fluorescence in situ hybridization method based on achieving super-resolution imaging according to any one of claims 1 to 10.

12. A method for detecting a disease caused by abnormal RNA expression, characterized in that: The method comprises the step of performing RNA signal localization based on the fluorescence in situ hybridization method for realizing super-resolution imaging as described in any one of claims 1 to 10.

13. The method for detecting a disease caused by abnormal RNA expression according to claim 12, characterized in that: The diseases caused by abnormal RNA expression include: diseases caused by abnormal MYC mRNA expression, diseases caused by abnormal TP53 mRNA expression, diseases caused by abnormal EGFR mRNA expression, diseases caused by abnormal BRAF mRNA expression, diseases caused by abnormal HER2 mRNA expression and / or diseases caused by abnormal SRC mRNA expression.

14. The method for detecting a disease caused by abnormal RNA expression according to claim 13, characterized in that: Diseases with abnormal MYC mRNA expression include lymphoma, lung cancer, breast cancer, gastric cancer, colorectal cancer, cervical cancer, leukemia, neuroblastoma, retinoblastoma, and sarcoma; Diseases with abnormal TP53 mRNA expression include breast cancer, lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer, hematological malignancies, and gliomas; Diseases with abnormal EGFR mRNA expression include non-small cell lung cancer, head and neck cancer, colorectal cancer, brain cancer, and pancreatic cancer; Diseases with abnormal BRAF mRNA expression include malignant melanoma, thyroid cancer, colorectal cancer, non-small cell lung cancer, pancreatic cancer, test tube cancer, esophageal cancer, breast cancer, and ovarian cancer; Diseases with abnormal HER2 mRNA expression include breast cancer and gastric cancer; Diseases with abnormal SRC mRNA expression include breast cancer, colorectal cancer, lung cancer, prostate cancer, gastric cancer, osteosarcoma, soft tissue sarcoma, renal cancer, familial macrognathia, and cardiovascular disease.