A method for detecting small RNA using mammalian embryo culture fluid

Abstract

The present invention is a method for extracting and detecting the type and expression of RNA, in particular, a method for extracting and detecting trace RNA in the culture fluid of cells. Various cells will secrete trace amounts of RNA molecules into the culture fluid during the culture process. This method is to add a linker to the 3' end and 5' end of the RNA molecule in the sample that may contain RNA, especially the culture fluid of the cell, and then perform a reverse transcription reaction, and then amplify. The amplified DNA product can also be analyzed by the second generation sequencing method to determine the type and expression of RNA secreted by the cell into the culture fluid. The extraction and amplification method of the present invention can be combined with sequencing and other methods to detect RNA, and the operation is simple, and the RNA information in the cell can be obtained without destroying the original cell.

Description

Technical Field

[0001] The invention relates to the fields of biomedicine and molecular cell biology, and in particular to a method for extracting and analyzing trace RNA in a sample. Background Art

[0002] Messenger RNA is the product of transcription using a strand of DNA as a template. The genetic information carried by messenger RNA can guide protein synthesis and contains a large amount of genetic information. Therefore, the sequence information of mRNA is very important for understanding gene expression. In addition to messenger RNA that guides protein synthesis, there are a large number of templates in the genome that transcribe non-coding RNA. These include various small RNA molecules (miRNA, siRNA, piwiRNA, piRNA, snoRNA, scaRNA, sdRNA, tsRNA, etc.), which are approximately 22-50nt in length. These RNAs affect a variety of life processes, including the process of embryonic development and the process of stem cell differentiation. More than half of the genes are regulated by various small RNA molecules. Small RNA molecules not only exist inside cells, but also release small RNA molecules into the culture medium through other methods such as exosomes. Since 1) messenger RNA contains a large amount of transcriptome information; 2) small RNA molecules have relatively good stability and relatively stable expression, RNA is a potential biomarker for non-invasive detection.

[0003] For example, non-invasive testing of IVF embryos is an important application area. In my country, one in eight couples encounters fertility difficulties, and couples who cannot have children are getting younger, with most patients aged 25 to 30. At present, there are more than 40 million infertile patients in China, accounting for 12.5% ​​of the childbearing population. In my country, there are about 3 million women of childbearing age who need assisted reproduction. And unfortunately, this number is still rising year by year. The huge demand for IVF has brought high requirements for IVF technology.

[0004] The process of IVF is ovulation induction, egg retrieval, sperm retrieval, in vitro fertilization, embryo culture, embryo transfer, corpus luteum support and pregnancy test. At present, the success rate of IVF is not high, and the success rate from embryo implantation to fetal birth is about 30%. A large part of the low success rate is due to unqualified fertilized eggs. Therefore, the quality of the fertilized egg is crucial. The most fundamental method to judge the quality of the fertilized egg is the morphological judgment method. Further genetic testing is achieved through invasive methods. The detection of in vitro fertilized eggs usually requires taking a cell out of the embryo and then sequencing the single cell. So far, scientists have found that the expression of some specific small molecule RNAs is related to the health of the embryo. However, such operations may have a negative impact on the healthy growth of the baby after birth. Not only that, the process of taking a single cell from the embryo requires precise operation under a microscope, which can be said to be a time-consuming and labor-intensive task. In order to solve this problem, people began to judge the health of the corresponding embryo by testing the embryo culture fluid, which is a non-invasive test for the embryo. Therefore, by measuring RNA in the culture medium, it is hoped that the accuracy of non-invasive testing will be increased. However, due to the limited amount of RNA released by the embryo, its success rate is still somewhat lower than that of single-cell testing.

[0005] For selecting RNA biomarkers, the most efficient method is to qualitatively and quantitatively determine which RNA is in the culture medium. However, existing RNA extraction and analysis technologies have many limitations: 1) a large amount of starting small RNA is required; 2) small RNA chains are very short, so it is difficult to obtain and amplify small RNA; 3) the efficiency of RNA plus adapter connection is too low; 4) only RNA with known sequences can be amplified. Therefore, it is urgent to find a technology to extract and analyze trace RNA in cell culture medium. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies in the above-mentioned prior art and provide a method for extracting and detecting trace RNA, especially a method for detecting using cell culture fluid, which can improve the success rate of obtaining RNA, especially small molecule RNA, and amplification, improve the efficiency of linker connection, and improve the detection efficiency of trace RNA. The method of the present invention is simple to operate and has higher safety and reliability.

[0007] In a first aspect of the present invention, a method for detecting RNA is provided, the method comprising the following steps:

[0008] (1) Providing samples that may contain RNA;

[0009] (2) adding lysis buffer and RNA digestion enzyme inhibitor;

[0010] (3) adding DNA digestion enzyme to digest the DNA in the sample;

[0011] (4) adding ribosomal RNA inhibitory primers;

[0012] (5) adding a 3' adapter primer and RNA ligase to connect to the 3' end of the RNA in the sample;

[0013] (6) Adding reverse transcription primer;

[0014] (7) adding a 5' adapter primer and RNA ligase to connect to the 5' end of the RNA in the sample;

[0015] (8) adding RNA digestion enzyme inhibitor and reverse transcriptase for reverse transcription;

[0016] (9) adding RNA digestion enzyme to digest RNA in the sample;

[0017] (10) adding amplification primers and DNA polymerase to carry out PCR amplification reaction;

[0018] (11) After the reaction in step 10 is completed, amplification primers and DNA polymerase are added to carry out a secondary PCR amplification reaction.

[0019] Preferably, the RNA-containing sample is a cell culture medium, more preferably a culture medium in which a fertilized egg is cultured for more than or equal to 24 hours before forming 8 cells or before the blastocyst stage.

[0020] Preferably, the RNA inhibitory primer is a 5.8s ribosomal RNA inhibitory primer, whose sequence is SEQ ID NO: 1, preferably SEQ ID NO: 6, ie, the 3' end of the primer contains a biotin modification of a TEG spacer, more preferably SEQ ID NO: 11.

[0021] In any of the above preferred embodiments, the above. Most preferably, the above.

[0022] In another preferred embodiment, the sequence of the 3' linker primer is SEQ ID NO: 3, more preferably, SEQ ID NO: 7, that is, the 5' end of the primer SEQ ID NO: 7 is adenylylated and the 3' end is dideoxycytidine modified.

[0023] In another preferred embodiment, the reverse transcription primer in step (6) has a sequence of SEQ ID NO: 8, ie, the 5' end is biotin-modified.

[0024] In another preferred embodiment, the 5' linker primer has a sequence of SEQ ID NO: 9, preferably SEQ ID NO: 10, and its 5' end contains an amino modification.

[0025] In another preferred embodiment, the RNA digestion enzyme in step (9) includes any one of RNase A and RNase T1 or a combination thereof.

[0026] In another preferred embodiment, the amplification primers in step (10) include a forward amplification primer and a reverse amplification primer. Preferably, the forward amplification primer sequence is SEQ ID NO: 3, and the reverse amplification primer sequence is SEQ ID NO: 4.

[0027] In another preferred embodiment, the procedure of the amplification reaction in step (10) is:

[0028]

[0029] In another preferred embodiment, the procedure of the secondary amplification reaction in step (11) is:

[0030]

[0031] In another preferred embodiment, the step (2) is to add 0.01%-0.2% Triton X-100 as a lysis solution and a recombinant RNA digestion enzyme inhibitor, and incubate at 37° C. for 5-60 minutes.

[0032] In another preferred embodiment, the step (3) is to add DNA digestion enzyme I and Tris-HCl and incubate at 37 degrees for 15-60 minutes.

[0033] In another preferred embodiment, the step (4) is to add a 5.8s ribosomal RNA inhibitory primer with a sequence of SEQ ID NO: 11 and incubate at 72° C. for 10-45 minutes.

[0034] In another preferred embodiment, the step (5) is to add the 3' linker primer of SEQ ID NO: 7, PEG 8000, T4 RNA ligase 2, truncated KQ, Tris-HCl, MgCl2, DTT, and incubate at 30°C for 3-8 hours; then add 2-5 units of recombinant RNA digestion enzyme inhibitor, and incubate at 4°C for 5-16 hours.

[0035] In another preferred embodiment, the step (6) is adding a reverse transcription primer of SEQ ID NO: 8 and incubating at 30° C. for 5-45 minutes; and / or, Lambda exonuclease, deadenylase, and incubating at 37° C. for 5-45 minutes.

[0036] In another preferred embodiment, the step (7) is to add a 5' linker primer of SEQ ID NO: 10, ATP, T4 RNA ligase, Tris HCl, MgCl2, DTT, and incubate at 37°C for 20-60 minutes.

[0037] In another preferred embodiment, the step (8) is to add Tris-HCl, KCl, DTT, dNTP, recombinant RNA digestion enzyme inhibitor, incubate at 42°C for 20-75 minutes; add reverse transcriptase, and incubate at 75°C for 5-30 minutes.

[0038] In another preferred embodiment, the step (9) is to add RNA digestion enzymes RNase A and RNase T1, Tris-HCl, and incubate at 37° C. for 5-60 minutes.

[0039] In another preferred embodiment, the step (10) is to add MgCl2, DNA polymerase, dNTP, the forward amplification primer sequence added is SEQ ID NO: 3, the reverse amplification primer sequence is SEQ ID NO: 4, and the mixed solution is placed in an amplification instrument for amplification reaction, and the amplification program is:

[0040]

[0041] The step (11) is to add after the reaction in step (10) is completed, suck out part of the original reaction solution and place it in a new container, add MgCl2, DNA polymerase, dNTP, the added amplification primers include forward amplification primers and reverse amplification primers, the forward amplification primer sequence is SEQ ID NO: 3, and the reverse amplification primer sequence is SEQ ID NO: 4; and / or place the mixed solution in an amplification instrument for amplification reaction, and the amplification program is:

[0042]

[0043]

[0044] In another preferred embodiment, the method may further include purifying and sequencing the samples obtained according to claims 1-10, and detecting the types and expression levels of RNA in the samples.

[0045] In a second aspect of the present invention, a kit for use in the above-mentioned RNA extraction and detection method is provided, comprising:

[0046] (1) one or more recombinant RNA digestion enzyme inhibitors; and / or

[0047] (2) DNA digestion enzyme I; and / or

[0048] (3) 5.8S ribosomal RNA inhibitory primer; and / or

[0049] (4) 3' adapter primer; and / or

[0050] (5) 5' adapter primer; and / or

[0051] (6) reverse transcription primer; and / or

[0052] (7) One or more RNA digesting enzymes.

[0053] The beneficial effects of the present invention are that it is convenient, simple, does not require the use of tissues or cells for detection, has high extraction and detection efficiency, and can detect small molecule RNAs that are not detected by conventional methods.

[0054] Experimental procedures

[0055] Obtaining cell culture medium: obtain fertilized eggs by the single sperm method, culture them for more than or equal to 24 hours before forming 8 cells or before the blastocyst stage, and aspirate a certain amount of culture medium, such as 9 μl culture medium, as the starting material for detection.

[0056] 3 μl of lysis buffer (0.01%-0.2% Triton X-100, 2-5 units of recombinant RNA digestion enzyme inhibitor, Takara or NEB) was added to 9 μl of culture medium and incubated at 37°C for 5-60 minutes.

[0057] After the reaction in step 2, add 0.01-1 unit DNAse I, NEB; 10-200 pmol Tris-HCl, and incubate at 37 degrees for 15-60 minutes.

[0058] After the reaction in step 3, add 2 μl of 5.8s ribosomal RNA inhibitory primer (2-8 pmol), such as SEQ ID NO: 1 or SEQ ID NO: 6, the 3' end of the primer may or may not contain biotin modification of the TEG spacer, and incubate at 72° C. for 10-45 minutes.

[0059] After the reaction in step 4, add 10-50 pmol of 3' adapter primer, such as SEQ ID NO: 2, whose sequence is 5'ATCTAATTCTCGNNNNNNATGC 3', or SEQ ID NO: 7, whose sequence is 5'rAppTCTAATTCTCGNNNNNNATGC-ddC 3', with adenylylation modification at the 5' end and dideoxycytidine modification at the 3' end; 0.1-0.2 μl PEG 8000; 10-100 units of T4 RNA ligase 2, truncated KQ, NEB; 50-100 pmol Tris-HCl; 10-50 pmol MgCl2; 1-10 pmol DTT; 2-5 units of recombinant RNA digestion enzyme inhibitor, Takara or NEB. Incubate at 30°C for 3-8 hours in the first step. Incubate at 4°C for 5-16 hours in the second step.

[0060] After the reaction in step 5, add 50-500 pmol of reverse transcription primer, such as SEQ ID NO: 8, whose sequence is 5'biotin-GCATNNNNNNCGAGAATTAGrA 3', with biotin modification at the 5' end; 1-5 units of Lambda exonuclease, NEB; 5-50 units of deadenylase, NEB. Incubate at 30°C for 5-45 minutes in the first step. Incubate at 37°C for 5-45 minutes in the second step.

[0061] After the reaction in step 6, add 20-100 pmol of 5' adapter primer, such as SEQ ID NO: 9 or SEQ ID NO: 10; 1-5 pmol ATP; 2-10 units of T4 RNA ligase, Thermo Fisher; 5-50 pmol Tris HCl; 1-10 pmol MgCl2; 0.1-2 pmol DTT. Incubate at 37°C for 20-60 minutes.

[0062] After the reaction in step 7, add 100-500 pmol Tris-HCl; 200-1500 pmol KCl; 20-100 pmol DTT; 1-10 pmol dNTP; 2-5 units of recombinant RNA digestion enzyme inhibitor; 50-500 units of reverse transcriptase, ThermoFisher, for reverse transcription. Incubate at 42°C for 20-75 minutes in the first step. Incubate at 75°C for 5-30 minutes in the second step.

[0063] After the reaction in step 8, add 0.01-1 unit RNase A, Thermo Fisher; 0.01-2 units RNase T1, Thermo Fisher; 10-200 pmol Tris-HCl, and incubate at 37°C for 5-60 minutes.

[0064] After the reaction in step 9, add 10-200 pmol MgCl2; 1-50 units of DNA polymerase; 1-20 pmol dNTP; 0.01-2 pmol amplification primers, which may include forward amplification primers and reverse amplification primers, such as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Place the mixed solution in an amplification instrument for amplification reaction, and the amplification program is:

[0065]

[0066] After the reaction in step 10 is completed, aspirate 1-5 μl of the original reaction solution and place it in a new test tube, then add 10-100 pmol MgCl2; 0.1-10 units of DNA polymerase; 1-20 pmol dNTP; 0.001-2 pmol amplification primer. Place the mixed solution in an amplification instrument for amplification reaction. The amplification program is as follows:

[0067]

[0068] The amplified product was purified according to conventional methods, and then subjected to second-generation sequencing to identify the types and expression levels of small RNA contained in the culture medium.

[0069] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 . Schematic diagram of the detection method of the present invention.

[0071] Figure 2 .The expression level of miR-372 was 55rpm.

[0072] Figure 3 .The expression level of miR-515 was 66rpm.

[0073] Figure 4 .The expression level of miR-603 was 82rpm.

[0074] Figure 5 .The expression level of miR-191 was 76rpm. DETAILED DESCRIPTION

[0075] The specific implementation of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0076] The experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0077] Unless otherwise specified in the examples of the present invention, the reagents, experimental instruments and methods used are as follows:

[0078] Cell culture: Conventional cell culture methods can be used. The method of blastocyst culture is to obtain fertilized eggs through intracytoplasmic sperm injection, culture them to the blastomere stage on the third day, and then transfer them to newly prepared blastocyst culture medium for blastocyst culture.

[0079] Main reagents and consumables: RNA digestion enzyme inhibitor, Takara or NEB; DNAse I, NEB; T4 RNA ligase 2, truncated KQ, NEB; Lambda exonuclease, NEB; deadenylase, NEB; T4 RNA ligase, Thermo Fisher or NEB; reverse transcriptase, Thermo Fisher; RNase A, Thermo Fisher; RNase T1, Thermo Fisher; DNA polymerase, NEB.

[0080] Main instruments: Centrifuge, Eppendorf 5424R, PCR thermal cycler, Eppendorf Nexus GX2

[0081] Illumina NovaSeq 6000, etc.

[0082] The reagents, equipment or services mentioned in the instructions can be purchased from commercial channels.

[0083] Example 1

[0084] The method of the present invention is described in Figure 1 :

[0085] Obtaining embryo culture medium: Fertilized eggs were obtained by the single sperm method, and 9 μl of the culture medium cultured for 24 hours before the formation of the blastocyst stage was aspirated as the starting material for the test;

[0086] Add 3 μl of lysis buffer (0.15% Triton X-100, 3 units of recombinant RNA digestion enzyme inhibitor, Takara) to 9 μl of culture medium.

[0087] After the reaction in step 2, add 0.5 μl (0.05 unit) DNAse I, NEB; 0.5 μl (20 pmol) Tris-HCl, and incubate at 37 degrees for 20 min.

[0088] After the reaction in step 3, add 2 μl of 5.8s ribosomal RNA inhibitory primer (4 pmol), such as SEQ ID NO: 1 or SEQ ID NO: 6, the 3' end of the primer may or may not contain biotin modification of the TEG spacer, and incubate at 72° C. for 30 minutes.

[0089] After the reaction in step 4, add 0.2 μl (20 pmol) 3' linker primer, such as SEQ ID NO: 2, whose sequence is 5'ATCTAATTCTCGNNNNNNATGC 3', or SEQ ID NO: 7, the primer 5' end is adenylylated and the 3' end is dideoxycytidine modified; 0.15 μl PEG 8000; 0.25 μl (50 units) T4 RNA ligase 2, truncated KQ, NEB; 0.4 μl (80 pmol) Tris-HCl; 0.6 μl (15 pmol) MgCl2; 0.3 μl (2 pmol) DTT; 0.1 μl (4 units) recombinant RNA digestion enzyme inhibitor, Takara. The first step is incubated at 30°C for 4 hours. The second step is incubated at 4°C for 12 hours.

[0090] After the reaction in step 5, add 2.2 μl (220 pmol) of reverse transcription primer, such as SEQ ID NO: 8, whose sequence is 5'biotin-GCATNNNNNNCGAGAATTAGrA 3', and the 5' end of the primer contains biotin modification; 0.6 μl (3 units) Lambda exonuclease, NEB; 0.2 μl (10 units) deadenylase, NEB. Incubate at 30°C for 15 minutes in the first step. Incubate at 37°C for 30 minutes in the second step.

[0091] After the reaction in step 6, add 0.5 μl (50 pmol) of 5' adapter primer, SEQ ID NO: 9 or SEQ ID NO: 10, with amino modification at the 5' end of the primer; 0.2 μl (2 pmol) of ATP; 0.5 μl (5 units) of T4 RNA ligase, Thermo Fisher; 0.2 μl (20 pmol) of Tris HCl; 0.4 μl (4 pmol) of MgCl2; 0.4 μl (0.4 pmol) of DTT. Incubate at 37°C for 60 minutes.

[0092] After the reaction in step 7, add 2 μl (200 pmol) Tris-HCl; 2 μl (800 pmol) KCl; 1 μl (50 pmol) DTT; 0.9 μl (3 pmol) dNTP; 0.1 μl (4 units) recombinant RNA digestion enzyme inhibitor; 1 μl (200 units) reverse transcriptase, Thermo Fisher, for reverse transcription. The first step is incubated at 42°C for 60 minutes. The second step is incubated at 75°C for 15 minutes.

[0093] After the reaction in step 8, 0.5 μl (0.05 unit) RNase A, Thermo Fisher; 0.5 μl (0.05 unit) RNase T1, Thermo Fisher; and 50 pmol Tris-HCl were added, and the mixture was incubated at 37° C. for 30 minutes.

[0094] After the reaction in step 9, add 10 μl (50 pmol) MgCl2; 0.5 μl (1 unit) DNA polymerase, ThermoFisher; 16 μl (16 pmol) dNTP; 8 μl (0.08 pmol) forward amplification primer. Place the mixture in an amplification instrument for amplification reaction. The amplification program is:

[0095]

[0096] After the reaction in step 10 is completed, 2 μl of the original reaction solution is aspirated and placed in a new test tube, and then 10 μl (50 pmol) MgCl2; 0.25 μl (0.5 unit) DNA polymerase, Thermo Fisher; 10 μl (20 pmol) dNTP; 2.5 μl (0.05 pmol) reverse amplification primer; 0.5 μl (0.005 pmol) forward amplification primer are added. The mixed solution is placed in an amplification instrument for amplification reaction. The amplification program is as follows:

[0097]

[0098] The amplified product was purified according to conventional methods, and then subjected to second-generation sequencing to identify the types and expression levels of small RNA contained in the culture medium.

[0099] The amplified products were purified using the Zymo Research library purification kit or the NEB library purification kit, and then subjected to second-generation sequencing using the PE150 strategy on an Illumina Novaseq sequencer to identify the types and expression levels of small RNA contained in the culture medium.

[0100] Conventional RNA extraction and detection method: provide a cell culture fluid sample, add lysis solution and RNA digestion enzyme inhibitor, add ribosomal RNA inhibitor primer; add 3' adapter primer and RNA ligase, add reverse transcription primer, add 5' adapter primer and RNA ligase, reverse transcription primer, and then perform PCR.

[0101] Experimental results:

[0102] Through the data analysis of the second generation sequencing, the experimental method detected the content and expression of 95 small RNA molecules in the culture medium, including various small RNA molecules such as miRNA, siRNA, piwiRNA, piRNA, snoRNA, scaRNA, sdRNA, tsRNA, etc. The following are examples of small RNA molecules detected, such as miR-372, miR-515, miR-603 and miR-191. The corresponding RNA cannot be detected without the method of the present invention. Using conventional RNA extraction and detection methods or single-cell RNA extraction and detection methods, only the content and expression of 5 small RNA molecules in the culture medium were detected, and miR-372, miR-515, miR-603 and miR-191 were not detected.

[0103] The expression level of miR-372 after optimization was undetectable before 55rpm optimization, see Figure 2 The expression level of miR-515 after optimization was 66rpm and could not be detected before optimization. Figure 3 The expression level of miR-603 after optimization was 82rpm. It was undetectable before optimization. Figure 4 The expression level of miR-191 after optimization was 76rpm and was undetectable before optimization. Figure 5 .

[0104] The sequences used in the present invention are shown in the following table:

[0105]

[0106] Note: "N" refers to any base, TEG-biotin refers to inter-arm biotin. NH2 refers to amino modification. rApp refers to adenylylation modification. ddC refers to dideoxycytidine modification. Biotin refers to biotin modification. "r" refers to RNA modification. NH2C6 refers to amino modification at C6. "H" refers to A, C or T base.

[0107] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Dingtan Technology (Beijing) Co., Ltd. <120> A method for detecting small RNA using mammalian embryo culture fluid <130> P2018-0613 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 76 <212> DNA <213> Artificial sequence <220> <223> 5.8S ribosomal RNA inhibitory primer <220> <221> misc_feature <222> (57)..(76) <223> n is a, c, g, t or u <400> 1 atcggcaagc gacgctcaga caggcgtagc cccgggagga acccggggcc gcaagtnnnn 60 nnnnnnnnnn nnnnnn 76 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> 3' adapter primer <220> <221> misc_feature <222> (13)..(18) <223> n is a, c, g, t or u <400> 2 atctaattct cgnnnnnnat gc 22 <210> 3 <211> 52 <212> DNA <213> Artificial sequence <220> <223> Forward amplification primer <400> 3 aatgatacgg cgaccaccga ctgacagtcg aagtcagtca gacagtccga cg 52 <210> 4 <211> 63 <212> DNA <213> Artificial sequence <220> <223> Reverse amplification primer <400> 4 caagcagaag acggcatacg agatattcat cgtgactgga gtgcatctga ctcgagaatt 60 aga 63 <210> 5 <211> 76 <212> DNA <213> Artificial sequence <220> <223> 5.8S ribosomal RNA inhibitory primer <400> 5 atcggcaagc gacgctcaga caggcgtagc cccggggaga acccggggcc gcaagtgcgt 60 tcgaagtgtc gatgat 76

Claims

1. A non-diagnostic and non-therapeutic method for extracting and detecting trace amounts of small molecule RNA, characterized in that: The method comprises the following steps: (1) Providing a sample that may contain RNA, wherein the sample is a cell culture medium, the cell culture medium is a culture medium in which a fertilized egg is cultured for more than or equal to 24 hours before the formation of 8 cells or before the blastocyst stage, and the RNA in the cell culture medium includes miRNA, the miRNA includes miR-372, miR-515, miR-603 and miR-191, and the RNA in the cell culture medium also includes one or more of siRNA, piwiRNA, piRNA, snoRNA, scaRNA, sdRNA, and tsRNA; (2) adding lysis buffer and RNA digestion enzyme inhibitor; (3) adding DNA digestion enzyme to digest the DNA in the sample; (4) adding a ribosomal RNA inhibitory primer, wherein the RNA inhibitory primer is a 5.8s ribosomal RNA inhibitory primer, whose sequence is SEQ ID NO: 1 or SEQ ID NO: 6; (5) adding a 3' adapter primer and RNA ligase to connect to the 3' end of the RNA in the sample, wherein the sequence of the 3' adapter primer is SEQ ID NO: 2 or SEQ ID NO: 7; (6) Adding reverse transcription primer; (7) adding a 5' adapter primer and RNA ligase to connect to the 5' end of the RNA in the sample, wherein the sequence of the 5' adapter primer is SEQ ID NO: 9 or SEQ ID NO: 10; (8) adding RNA digestion enzyme inhibitor and reverse transcriptase for reverse transcription; (9) adding RNA digestion enzyme to digest RNA in the sample; (10) adding a forward amplification primer and a DNA polymerase to carry out a PCR amplification reaction; (11) After the reaction in step 10 is completed, a reverse amplification primer, a forward amplification primer and a DNA polymerase are added to carry out a secondary PCR amplification reaction.

2. The method according to claim 1, characterized in that The cell culture medium is the culture medium in which the fertilized egg is cultured for 24 hours before forming the blastocyst stage.

3. The method according to claim 1, characterized in that The sequence of the 5.8s ribosomal RNA inhibitory primer is SEQ ID NO:

1.

4. The method according to claim 1, characterized in that The sequence of the 5.8s ribosomal RNA inhibitory primer is SEQ ID NO: 6, and the 3' end of the primer contains a biotin modification of a TEG spacer.

5. The method according to claim 4, characterized in that The sequence of the 5.8s ribosomal RNA inhibitory primer is SEQ ID NO:

11.

6. The method according to claim 1, characterized in that The sequence of the 3' linker primer is SEQ ID NO:

2.

7. The method according to claim 1, characterized in that The sequence of the 3' linker primer is SEQ ID NO: 7, the 5' end of which is adenylylated and the 3' end is dideoxycytidine modified.

8. The method according to claim 1, characterized in that The reverse transcription primer in step (6) has a sequence of SEQ ID NO: 8, and its 5' end is modified with biotin.

9. The method according to claim 1, characterized in that The sequence of the 5' linker primer is SEQ ID NO:

9.

10. The method according to claim 1, characterized in that The sequence of the 5' linker primer is SEQ ID NO: 10, and the 5' end thereof contains an amino modification.

11. The method according to claim 1, characterized in that The RNA digestion enzyme in step (9) includes any one of RNase A and RNase T1 or a combination thereof.

12. The method according to claim 1, characterized in that The forward amplification primer sequence of step (10) is SEQ ID NO:

3.

13. The method according to claim 1, characterized in that The forward amplification primer sequence of step (11) is SEQ ID NO: 3, and the reverse amplification primer sequence is SEQ ID NO:

4.

14. The method according to claim 1, wherein: The procedure of the amplification reaction in step (10) is:

15. The method according to claim 1, wherein: The procedure of the secondary amplification reaction in step (11) is:

16. The method according to claim 1, wherein: The step (2) is to add 0.01%-0.2% TritonX-100 as a lysis solution and a recombinant RNA digestion enzyme inhibitor, and incubate at 37°C for 5-60 minutes; and the step (3) is to add DNA digestion enzyme I and Tris-HCl, and incubate at 37°C for 15-60 minutes; the step (4) is to add a 5.8s ribosomal RNA inhibitor primer with a sequence of SEQ ID NO: 11, and incubate at 72°C for 10-45 minutes; and the step (5) is to add a 3' linker primer of SEQ ID NO: 7, PEG 8000, T4 RNA ligase 2, truncated KQ, Tris-HCl, MgCl2, DTT, and incubate at 30°C for 3-8 hours; then add 2-5 units of recombinant RNA digestion enzyme inhibitor, and incubate at 4°C for 5-16 hours; and the step (6) is to add SEQ ID NO: IDNO: 8 reverse transcription primer, incubated at 30°C for 5-45 minutes; Lambda exonuclease, deadenylase, incubated at 37°C for 5-45 minutes; and the step (7) is to add the 5' linker primer of SEQ ID NO: 10, ATP, T4 RNA ligase, Tris HCl, MgCl2, DTT, incubated at 37°C for 20-60 minutes; and the step (8) is to add Tris-HCl, KCl, DTT, dNTP, recombinant RNA digestion enzyme inhibitor, incubated at 42°C for 20-75 minutes; add reverse transcriptase, incubated at 75°C for 5-30 minutes; and the step (9) is to add RNA digestion enzymes RNase A and RNase T1, Tris-HCl, incubated at 37°C for 5-60 minutes; and the step (10) is to add MgCl2, DNA polymerase, dNTP, and the added forward amplification primer sequence is SEQ ID NO:

3. The mixed solution is placed in an amplification instrument for amplification reaction, and the amplification program is: The step (11) is to add after the reaction in step (10) is completed, suck out part of the original reaction solution and place it in a new container, add MgCl2, DNA polymerase, dNTP, the added amplification primers include forward amplification primers and reverse amplification primers, the forward amplification primer sequence is SEQ ID NO: 3, and the reverse amplification primer sequence is SEQ ID NO: 4; and / or place the mixed solution in an amplification instrument for amplification reaction, and the amplification program is:

17. The method according to any one of claims 1 to 15, characterized in that: The method may further include purifying and sequencing the sample obtained in step (11), and detecting the type and expression level of RNA in the sample.

18. A kit for use in the extraction and detection method according to claim 1, characterized in that: The kit comprises: (1) one or more recombinant RNA digestion enzyme inhibitors; (2) DNA digestion enzyme I; (3) a 5.8s ribosomal RNA inhibitory primer, wherein the sequence of the 5.8s ribosomal RNA inhibitory primer is SEQ ID NO: 1 or SEQ ID NO: 6; (4) a 3' linker primer, wherein the sequence of the 3' linker primer is SEQ ID NO: 2 or SEQ ID NO: 7; (5) a 5' linker primer, wherein the sequence of the 5' linker primer is SEQ ID NO: 9 or SEQ ID NO: 10; (6) reverse transcription primer; and (7) one or more RNA digesting enzymes; and (8) Forward amplification primer and reverse amplification primer.

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