CN108642175B - Early embryo diapause villus tissue miRNAs biomarker and detection method of expression quantity thereof - Google Patents
Early embryo diapause villus tissue miRNAs biomarker and detection method of expression quantity thereof Download PDFInfo
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Abstract
The invention discloses early embryo diapause villus tissue miRNAs biomarkers related to early embryo diapause degree, which comprise hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5 p. Accordingly, the inventors have also designed corresponding reverse transcription or real-time fluorescent PCR primers targeting the above miRNAs biomarkers. The compounds can be used as biomarkers for detecting early embryo diapause by villus samples, or used for preparing tools such as diagnostic reagents or biochips, and the like, have important potential value and application prospect for diagnosis and treatment of early embryo diapause, can provide theoretical basis for the later research of diagnosing human early embryo diapause, tissues and serum miRNAs from a molecular level, and have important theoretical significance and potential practical value.
Description
Technical Field
The invention belongs to the technical field of biomarkers in biomedicine, and particularly relates to an early embryo diapause villus tissue miRNAs biomarker and a detection method of an expression level thereof.
Background
Early Embryo Arrest (EEA) is one of the major causes of female infertility. To date, the cause of early embryo diapause is unknown, and the treatment method is limited, which seriously affects the physical and mental health of women of childbearing age.
MicroRNAs (miRNAs) are important regulatory molecules in modern biomedicine, regulate and control various functions of all cells, tissues and organs of a human body through regulating and controlling gene transcription expression, and play an important role in the occurrence, prevention and treatment of human diseases. Numerous studies have found that miRNAs can be found widely and stably in extracellular fluids, including serum, plasma and interstitial fluid, not only as regulatory molecules of transcription and expression of intracellular genes, but also as signaling molecules for information transfer between cells. During the pathological process, the expression profiles of miRNAs in tissue fluids are characteristically altered, which contributes to the diagnosis and prognosis of the disease, even in close relation to clinical staging.
In normal functional states, circulating miRNAs species and their abundance of expression in the body may be in a relatively stable, balanced state. The variable, repetitive damage process that occurs during the onset of early embryonic diapause may directly or indirectly disrupt villous tissue miRNAs homeostasis, manifested by abnormal metabolism or significant increases or decreases in the expression levels of certain tissue miRNAs in specific disease states. There are no published reports on miRNAs of early embryo diapause villous tissues.
Disclosure of Invention
The invention aims to provide a detection method of early embryo diapause villus tissue miRNAs biomarkers and an expression level thereof, and provides support for accurate diagnosis of clinical early embryo diapause.
In order to solve the technical problems, the invention adopts the technical scheme that:
early embryo shutdown villus tissue miRNAs biomarkers, which comprise hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5p, and the biomarkers respectively have base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 6.
The miRNAs biomarkers of the early embryo diapause villus tissue are derived from human villus tissue.
Application of miRNAs biomarkers of early embryo diapause villus tissues in preparation of reagents, kits or biochips for diagnosing or assisting in diagnosing early embryo diapause.
The application of the miRNAs biomarkers of the early embryo diapause villus tissue in preparing or screening medicaments for treating early embryo diapause.
The primer designed by taking the early embryo diapause villus tissue miRNAs biomarker as a target comprises a base sequence from a sequence table SEQ.ID.No.7 to a sequence table SEQ.ID.No. 14.
The primer is applied to the preparation of a reagent, a kit or a biochip for diagnosing or assisting in diagnosing early embryo diapause.
The application of the primer in preparing or screening the medicament for treating early embryo diapause.
The method for detecting the expression level of the early embryo diapause villus tissue miRNAs biomarkers comprises the following steps:
(1) total RNA extraction
Adding liquid nitrogen into villus tissue, grinding into powder, putting 50-100mg of the ground powder into a 2mL centrifuge tube, simultaneously adding 1mL TRIZOL to fully crack the villus tissue, standing at room temperature for 5 minutes, adding 200 mu L chloroform, mixing uniformly by vortex for 15 seconds, and standing on ice for 15 minutes; centrifuging at 12000g at 4 ℃ for 15 minutes; absorbing the upper-layer water phase, adding precooled isopropanol with the same volume, gently mixing uniformly, and standing for 10 minutes on ice; centrifuging at 12000g at 4 ℃ for 10 minutes, and removing supernatant; adding 10mL of 75% ethanol prepared from precooled DEPC-treated water, carefully resuspending the precipitate, centrifuging at 12000g at 4 ℃ for 10 minutes, and discarding the supernatant; washing the precipitate repeatedly; sucking out the supernatant, and placing the supernatant on a super-clean workbench for air drying and precipitating for 5 to 10 minutes; finally, 20 μ L of double distilled water treated with DEPC was added;
(2) removal of genomic DNA from Total RNA
The method is carried out by adopting a kit for removing genomic DNA, wherein a reaction system comprises 0.4 mu L of 2U mu L DNase I, 2 mu L of 10-fold reaction buffer solution and 0.6 mu L of 20U mu L of LRiboLock RNase inhibitor, 4 mu g of total RNA is added into each reaction system, and the total reaction system is 20 mu L; the reaction conditions are as follows: reacting at 37 ℃ for 30 minutes, and then inactivating at 75 ℃ for 10 minutes;
(3) reverse transcription
Carrying out reverse transcription reaction on the miRNAs by adopting a reverse transcription kit and a miRNAs specific stem-loop structure reverse transcription random primer; wherein the reaction system comprises 0.5 muL of reverse transcription random primer, 2 muL of 5-star reverse transcription buffer solution, 1 muL of 10mmol/L dNTP mixture, 0.5 muL of 20U muLRiboLock RNase inhibitor and 0.5 muL of 200U muL reverse transcriptase, 1 mug of RNA with gene DNA removed is added into each reaction system, and the total reaction system is 10 muL; the reaction conditions are that the mixture is incubated at 65 ℃ for 5 minutes and immediately placed on ice for 5 minutes, then reacted at 42 ℃ for 60 minutes, and finally incubated at 70 ℃ for 5 minutes;
(4) real-time fluorescent quantitative PCR
5 uL 2 × SYBR Green mixed solution, 0.05 uLROX, 1.4 uLmiRNAs primer mixed solution, 1 uL diluted cDNA, 2.55 uL double distilled water, 10 uL total reaction system, and each miRNA detection is implemented by 4 parallel repeats; the rapid real-time fluorescent quantitative PCR instrument is used for detection, and the reaction conditions of the operation of the instrument are as follows: 40 cycles of 95 ℃ for 2 minutes, 95 ℃ for 5 seconds, and 60 ℃ for 30 seconds were performed.
The reverse transcription random primer in the step (3) has a base sequence of a sequence table SEQ ID No.14, and the primer mixture in the step (4) includes base sequences of sequence tables SEQ ID No.7 to SEQ ID No. 13.
The inventor adopts a high-throughput sequencing and real-time quantitative PCR method for detection, and finds that early embryo shutdown villus tissue miRNAs biomarkers which have high expression or low expression in tissues of patients with early embryo shutdown and are related to the degree of early embryo shutdown comprise hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5p, and the biomarkers respectively have base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 6. Accordingly, the inventors have also designed corresponding reverse transcription or real-time fluorescent PCR primers comprising the base sequences of the sequence listing seq.id No.7 to seq.id No.14, targeting the above miRNAs biomarkers. Experiments prove that hsa-miR-136-5p, hsa-miR-1301-3p, hsa-141-3p and hsa-miR-135b-5p are highly expressed in the villus tissue of the early embryo diapause patient, hsa-miR-19b-3p and hsa-miR-486-5p are lowly expressed in the villus tissue of the early embryo diapause patient, therefore, they can be used as a villus sample to detect biomarkers of early embryo diapause, or used for preparing tools such as diagnostic reagents or biochips, the method has important potential value and application prospect for diagnosis and treatment of early embryo diapause, can provide theoretical basis for research on diagnosis of early embryo diapause, tissues and serum miRNAs from molecular level in the future, and has great theoretical significance and potential practical value.
Drawings
FIG. 1 is a histogram of the expression of has-miR-1301-3p in the control group and the experimental group detected by qPCR.
FIG. 2 is a histogram of the expression of has-miR-141-3p in the control group and the experimental group measured by qPCR.
FIG. 3 is a histogram of the expression of has-miR-19b-3p in the control group and the experimental group measured by qPCR.
FIG. 4 is a histogram of the expression of has-miR-135b-5p in the control group and the experimental group measured by qPCR.
FIG. 5 is a histogram of expression of has-miR-486-5p in control and experimental groups measured by qPCR.
FIG. 6 is a histogram of the expression of has-miR-136-5p in the control group and the experimental group measured by qPCR.
In the figure: the abscissa represents the control group and the experimental group, respectively, and the ordinate represents the average expression level.
Detailed Description
The present invention will be described in detail below by way of examples with reference to the accompanying drawings, in which conditions and methods not specified are performed according to conventional conditions.
In the embodiment, the miRNAs biomarkers of the villus tissue of early embryo diapause are used for detecting early embryo diapause, and the specific process comprises the following steps:
1. screening candidate early embryo diapause related miRNAs by adopting microRNAs high-throughput sequencing:
villus tissues of 3 normal early pregnancy healthy controls and 3 early embryo diapause patients are collected, total RNA is extracted by using a Trizol method, and quality inspection is carried out on the obtained total RNA, so that the concentration and the integrity of the total RNA can meet the sequencing requirement. 1.5 mu g of total RNA is taken from each Sample, 18-32nt fragments are enriched by using a Small RNA enrichment kit, a Small RNA library is constructed by using a Small RNA Sample PrepKit kit, joint primers of the purified Small RNA are respectively connected to the 5 'end and the 3' end of the sequences of the two libraries by using T4RNA ligase, a first cDNA chain is synthesized by reverse transcription, and a Small RNA library is established by using a common PCR method. The library was subjected to high throughput sequencing using the Illumina HiSeq 2500 high throughput sequencing platform. The small RNA sequence with the length of 51nt obtained by sequencing is subjected to joint removal and low-quality and pollution sequence removal, and finally a high-quality small RNA sequence with the length of 18-32nt is obtained. And comparing and analyzing the obtained small RNA sequence with a reference genome, comparing the matched sequences with sequences in a database respectively, screening miRNA sequences, carrying out classified annotation, and analyzing the expression condition of the miRNA sequences. Standardizing the expression quantity of miRNAs in a sample into TPM (tags per million), analyzing and counting the expression differences of the known miRNAs in 2 groups of samples, screening differential miRNAs by utilizing a P value and a fold change value of T test according to preset conditions, and primarily screening the miRNAs specifically expressed in the villus tissue of an early embryo diapause patient, wherein the screening standard is as follows:
1) fold Change in expression level (Fold Change) ≥ 2 (low expression < 0.5);
2) the P value is less than or equal to 0.01;
screening 36 candidate miRNAs, wherein the expression of 22 miRNAs is up-regulated, and the expression is respectively as follows: hsa-miR-549a, miR-200-x, miR-6236-y, miR-3940-y, hsa-miR-2276-3p, miR-8304-y, hsa-miR-4800-3p, miR-4661-x, hsa-miR-589-3p, hsa-miR-4664-5p, hsa-miR-556-3p, novel-m0066-5p, novel-m0045-5p, hsa-miR-136-5p, hsa-miR-3614-3p, hsa-miR-1301-3p, hsa-miR-24-1-5p, miR-24-x, hsa-miR-141-3p, hsa-miR-3614-5p, hsa-miR-3914-3 p, hsa-miR-2116-3p and hsa-miR-135b-5 p; the expression of 14 miRNAs was down-regulated, respectively: miR-126-x, miR-4483-y, hsa-miR-33a-5p, miR-539-y, miR-17-x, hsa-miR-19b-3p, miR-19-y, miR-142-y, hsa-miR-144-5p, hsa-miR-486-5p, hsa-miR-16-1-3p, miR-144-x, miR-486-x and hsa-miR-144-rp.
2. Real-time fluorescent quantitative PCR (polymerase chain reaction) verification of miRNAs (micro ribonucleic acids) in villus tissues of patients with early embryo arrest
4 miRNAs are up-regulated in expression amount (hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p and hsa-141-3p) and 2 miRNAs are down-regulated in expression amount (hsa-miR-19b-3p and hsa-miR-486-5p) in villus tissue of a patient with early embryo abortion, and the biomarker for diagnosing early embryo abortion is subjected to next real-time fluorescent quantitative PCR (polymerase chain reaction) verification analysis. Villous tissue from 6 patients with early embryo diapause was collected as a test group, while 6 age and gender matched normal early pregnancy healthy controls were collected (table 1). The specific detection process is as follows:
(1) total RNA extraction: adding a proper amount of villus tissue into liquid nitrogen, quickly grinding into powder, putting 50-100mg of the ground powder into a 2mL centrifuge tube, simultaneously adding 1mL TRIZOL to fully crack the villus tissue, standing at room temperature for 5 minutes, adding 200 mu L chloroform, whirling and uniformly mixing for 15 seconds, and standing on ice for 15 minutes; centrifuging at 12000g at 4 ℃ for 15 minutes; carefully absorbing the upper aqueous phase, adding precooled isopropanol with the same volume, gently mixing uniformly, and standing for 10 minutes on ice; centrifuging at 12000g at 4 ℃ for 10 minutes, and removing supernatant; adding 10mL of 75% ethanol prepared from precooled DEPC-treated water, carefully resuspending the precipitate, centrifuging at 12000g at 4 ℃ for 10 minutes, and discarding the supernatant; washing the precipitate repeatedly; the supernatant is fully sucked out and is placed in a super clean bench for air drying precipitation (5-10 minutes), the precipitation is not suitable for being blown to be dried, and the RNA is prevented from being dried and not easy to dissolve; finally, 20. mu.L of double distilled water treated with DEPC was added.
(2) Removal of genomic DNA from total RNA: the method is carried out by using a kit for removing genomic DNA, the reaction system comprises 0.4 mu L of DNase I (2U/. mu.L), 2 mu L of 10-th reaction buffer and 0.6 mu L of RiboLock RNase inhibitor (20U/. mu.L), 4 mu g of total RNA is added into each reaction system, and the total reaction system is 20 mu L. The reaction conditions of the method are as follows: the reaction was carried out at 37 ℃ for 30 minutes and then inactivated at 75 ℃ for 10 minutes.
(3) Reverse transcription: and carrying out reverse transcription reaction on the miRNAs by adopting a reverse transcription kit and a miRNAs specific stem-loop structure reverse transcription random primer. Wherein the reaction system comprises 0.5 mu L of reverse transcription random primer, 2 mu L of 5-th reverse transcription buffer solution, 1 mu L of 10mmol/L dNTP mixture, 0.5 mu L of RiboLock RNase inhibitor (20U/. mu.L) and 0.5 mu L of reverse transcriptase (200U/. mu.L), 1 mu g of RNA with gene DNA removed is added into each reaction system, and the total reaction system is 10 mu L. The reaction conditions of this method were 65 ℃ incubation for 5 minutes immediately on ice for 5 minutes, then 42 ℃ reaction for 60 minutes, and finally 70 ℃ incubation for 5 minutes.
(4) Real-time fluorescent quantitative PCR: 5 uL of 2 × SYBR Green mixture, 0.05 uL ROX (instrument fluorescence correction fluid), 1.4 uL of miRNAs primer mixture, 1 uL of diluted cDNA and 2.55 uL of double distilled water, wherein the total reaction system is 10 uL, and each miRNA detection is carried out by 4 parallel repeats. The ABI 7500 type rapid real-time fluorescence quantitative PCR instrument is used for detection, and the reaction conditions of the instrument operation are as follows: the detection of the expression level of the early embryo diapause villus tissue miRNAs biomarkers is completed by 40 cycles of 95 ℃ for 2 minutes, 95 ℃ for 5 seconds and 60 ℃ for 30 seconds (the specific primers are shown in Table 3).
(5) In real-time fluorescent quantitative PCR experiments, the Ct value is used as a relative quantitative parameter for miRNAs as a result, and the principle is that the Ct value is in linear inverse proportion to the logarithm of the copy number of the initial template in the sample. Wherein "C" in Ct represents Cycle and "t" represents threshold for detecting threshold, which means the number of cycles required for the fluorescence signal intensity to reach the threshold during PCR amplification. In the test, when the Ct value is more than 35, the miRNA expression in the sample is considered to be low and cannot be detected or does not contain the miRNA; when the target miRNAs have the same amplification efficiency as the internal reference gene, the amplification efficiency can be determined by taking the value of the target gene Ct-the value of the internal reference gene Ct,
the Δ Δ Ct is the Δ Ct value of each case sample-the Δ Ct average value of the control group, and the Δ Ct in this test is the target miRNA Ct-the internal reference Ct, thereby obtaining the quantification of the target miRNA relative to the internal reference. By using 2-ΔΔctCalculating the change multiple of the expression quantity of the miRNA in the experimental group relative to the change multiple of the miRNA in the control group, establishing a negative control and a repeated test in the test, and repeating all real-time fluorescence quantitative PCR for 3 times; the result data are statistically analyzed by using SPSS 17.0 software, and the differential expression level of each miRNA between two groups is counted by using a Nonpaametric Mann-Whitney test method, and the difference is statistically considered to have statistical significance when P is less than 0.05. The data processing results of the miRNAs with differential expression are all expressed by mean value + standard error, and simultaneously, a column diagram containing error lines is drawn; the results are shown in FIG. 1-FIG. 6, where hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p and hsa-141-3p are highly expressed in the villus tissue of patients with early embryonic arrest, and hsa-miR-19b-3p and hsa-miR-486-5p are expressed in the early embryonic stageThe expression is low during the birth, which is consistent with the result of primary screening.
TABLE 112 tissue samples
TABLE 26 sequences as biomarkers for miRNAs for diagnosis of early embryo diapause
| Names of miRNAs | Sequence of |
| has-miR-136-5p | acuccauuuguuuugaugaugga |
| has-miR-1301-3p | uugcagcugccugggagugacuuc |
| has-miR-135b-5p | uauggcuuuucauuccuauguga |
| has-miR-141-3p | uaacacugucugguaaagaugg |
| has-miR-19b-3p | gugcaaauccaugcaaaacuga |
| has-miR-486-5p | uccuguacugagcugccccgag |
TABLE 3 real-time fluorescent quantitative PCR primer information
In conclusion, the invention establishes a screening method of the tissue miRNAs biomarkers and an expression profile of early embryo diapause villus tissue miRNAs, finds that the miRNAs in the villus tissue of an early embryo diapause patient are abnormally expressed, and discloses the value of the villus tissue miRNAs on early embryo diapause detection. Wherein, hsa-miR-136-5p, hsa-miR-1301-3p, hsa-141-3p and hsa-miR-135b-5p are highly expressed in tissues of patients with early embryonic diapause, hsa-miR-19b-3p and hsa-miR-486-5p are lowly expressed in tissues of patients with early embryonic diapause, and can be miRNAs molecules which are characteristic of the patients with early embryonic diapause, and can be used as markers for diagnosis or auxiliary diagnosis and drugs for screening and treating early embryonic diapause. Wherein, hsa-miR-136-5p, hsa-141-3p, hsa-miR-1301-3p and hsa-miR-135b-5p are up-regulation expression markers, and hsa-miR-19b-3p and hsa-iR-486-5p are down-regulation expression markers, which can be used as early embryo diapause diagnosis markers or auxiliary diagnosis markers in villus tissue samples. The miRNAs can be used as a diagnostic marker of early embryo diapause, can be used for auxiliary diagnosis of patients with early embryo diapause, has the advantages of small detection damage, good stability, high sensitivity and the like, and provides support for accurate diagnosis of early embryo diapause clinically. The method has important academic significance and application prospect for auxiliary diagnosis of early embryo diapause, enrichment of pathological mechanisms of early embryo diapause and the like.
Sequence listing
<110> Guangxi medical university first subsidiary hospital
NANNING WILKING BIOLOGICAL TECHNOLOGY Co.,Ltd.
<120> early embryo diapause villus tissue miRNAs biomarker and detection method of expression quantity thereof
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acuccauuug uuuugaugau gga 23
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uugcagcugc cugggaguga cuuc 24
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gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacnnnnnn 50
Claims (6)
1. The miRNAs biomarkers of early embryo shutdown villus tissues are characterized by comprising hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5p which are respectively the base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 6.
2. Use of the early embryo diapause villus tissue miRNAs biomarkers of claim 1 in the preparation of a reagent, kit or biochip for diagnosis or assisted diagnosis of early embryo diapause.
3. A primer designed with the early embryo diapause villus tissue miRNAs biomarker as described in claim 1, which is characterized by comprising the base sequence of SEQ ID No.7 to SEQ ID No. 14.
4. Use of the primer of claim 3 for the preparation of a reagent, a kit or a biochip for the diagnosis or the auxiliary diagnosis of early embryo diapause.
5. The method for the non-diagnostic detection of the expression level of early embryo diapause villus tissue miRNAs biomarkers of claim 1, comprising the steps of:
(1) total RNA extraction
Adding liquid nitrogen into villus tissue, grinding into powder, putting 50-100mg of the ground powder into a 2mL centrifuge tube, simultaneously adding 1mL TRIZOL to fully crack the villus tissue, standing at room temperature for 5 minutes, adding 200 mu L chloroform, mixing uniformly by vortex for 15 seconds, and standing on ice for 15 minutes; centrifuging at 12000g at 4 ℃ for 15 minutes; absorbing the upper-layer water phase, adding precooled iso-propanol, lightly mixing uniformly, and standing on ice for 10 minutes; centrifuging at 12000g at 4 ℃ for 10 minutes, and removing supernatant; adding 10mL of 75% ethanol prepared from precooled DEPC-treated water, carefully resuspending the precipitate, centrifuging at 12000g at 4 ℃ for 10 minutes, and discarding the supernatant; washing the precipitate repeatedly; sucking out the supernatant, and placing the supernatant on a super-clean workbench for air drying and precipitating for 5 to 10 minutes; finally, 20 μ L of double distilled water treated with DEPC was added;
(2) removal of genomic DNA from Total RNA
The method is carried out by adopting a kit for removing genome DNA, a reaction system comprises 0.4 mu L of 2U/. mu.L DNase I, 2 mu L of 10-inch reaction buffer solution and 0.6 mu L of 20U/. mu.L RiboLock RNase inhibitor, 4 mu g of total RNA is added into each reaction system, and the total reaction system is 20 mu L; the reaction conditions are as follows: reacting at 37 ℃ for 30 minutes, and then inactivating at 75 ℃ for 10 minutes;
(3) reverse transcription
Carrying out reverse transcription reaction on the miRNAs by adopting a reverse transcription kit and a miRNAs specific stem-loop structure reverse transcription random primer; wherein the reaction system comprises 0.5 muL of reverse transcription random primer, 2 muL of 5-prime reverse transcription buffer solution, 1 muL of 10mmol/L dNTP mixture, 0.5 muL of 20U/muL RiboLock RNase inhibitor and 0.5 muL of 200U/muL reverse transcriptase, 1 mug of RNA with gene DNA removed is added into each reaction system, and the total reaction system is 10 muL; the reaction conditions are that the mixture is incubated at 65 ℃ for 5 minutes and immediately placed on ice for 5 minutes, then reacted at 42 ℃ for 60 minutes, and finally incubated at 70 ℃ for 5 minutes;
(4) real-time fluorescent quantitative PCR
5 μ L of 2 SYBR Green mixed liquor, 0.05 μ L ROX, 1.4 μ L miRNAs primer mixed liquor, 1 μ L diluted cDNA, 2.55 μ L double distilled water, 10 μ L total reaction system, and 4 parallel repeats of each miRNA detection; the rapid real-time fluorescence quantitative PCR instrument is used for detection, and the reaction conditions of the instrument operation are as follows: 40 cycles of 95 ℃ for 2 minutes, 95 ℃ for 5 seconds, and 60 ℃ for 30 seconds were performed.
6. The method for detecting the expression level of miRNAs biomarkers in early embryo diapauzing villus tissue without diagnostic purpose as claimed in claim 5, wherein: the reverse transcription random primer in the step (3) is a base sequence of a sequence table SEQ ID No.14, and the primer mixed solution in the step (4) comprises base sequences of sequence tables SEQ ID No.7 to SEQ ID No. 13.
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