Method for identifying menstrual blood and peripheral blood by using expression quantity relationship of miRNA based on RNU6b as reference gene

Abstract

The invention discloses a method for identifying menstrual blood and peripheral blood by using an expression quantity relationship of miRNA based on RNU6b as a reference gene, and relates to the technical field of biology. According to the invention, three miRNAs (miR-144-5p, miR-203-3p and miR-451a) with high expression difference are screened from vaginal blood and peripheral blood on the basis of a high-throughput sequencing technology for the first time, and the difference of screening indexes is further verified in other three body fluids (semen, saliva and vaginal secretion); meanwhile, six reference genes (RNU6b, U6, miR-103a-3p, miR-191-5p, miR-92a-3p and miR-484) are screened to evaluate comparison results, and the RNU6b with the highest stability is finally selected as the reference gene, so that an efficient and accurate identification method system is finally established, or the method will become a powerful tool for body fluid plaque tissue sources of forensic physical evidence, and can be used as a kit for identifying body fluid plaque tissue sources of forensic physical evidence. And more scientific and reasonable auxiliary evidences are provided for qualitative and detection of cases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to identifying menstrual blood and peripheral blood based on the expression relationship of miRNA using RNU6b as an internal reference gene. Background Art

[0002] Traditional methods for distinguishing peripheral blood from menstrual blood include fibrinolytic system activity detection, microscopic genital tract cell detection, lactate dehydrogenase (LDH) isozyme detection, etc. These methods are highly subjective and the results obtained are often inaccurate. In recent years, the development of molecular biology and genetics has provided a series of new ideas for the identification of human body fluids and tissues, such as using the tissue-specific expression characteristics of messenger RNA (mRNA), DNA methylation or non-coding RNA (ncRNA) to achieve the purpose of specifically testing body fluid stains. At present, the positive identification of several common body fluids in forensic medicine (semen, urine, peripheral blood, saliva, vaginal secretions and menstrual blood, etc.) can only be achieved through the collection detection of RNA biomarkers. Although both mRNA and DNA methylation detection methods have been applied in actual cases, these two technologies also have certain limitations. DNA methylation and microbial methods can identify fewer types of body fluids, and mRNA chains are long, and their degradation is inevitable over time, which may affect the accuracy of the test and identification results. Therefore, miRNA has entered the field of vision of researchers and has become the focus of forensic researchers in the identification of the tissue source of body fluid stains. As a type of single-stranded endogenous non-coding small RNA molecule, miRNA is about 20-25 nucleotides in size. Its expression is highly tissue-specific and temporal, and it is not easily degraded and has strong stability. These advantages provide a basis for the application of miRNA in forensic work, making it of great significance and good application prospects in forensic evidence detection. At the same time, although miRNA has strong stability, in theory, it is still affected by RNAse. Previous clinical studies usually stored blood samples under freezing conditions to study the stability of miRNA in order to find its optimal storage conditions. "Tissue-specific miRNA" should strictly be expressed as "cell-specific miRNA". The specific miRNA expressed by differentially expressed cells with abundant content is the body fluid tissue-specific miRNA. From the perspective of "cell-specific miRNA", the reasons for the difficulty in identifying blood samples and the feasible identification points are: the difference between blood samples and non-blood samples is that blood samples contain a large number of blood cells, and although both peripheral blood and menstrual blood contain a large number of red blood cells, compared with peripheral blood, menstrual blood also contains a large number of endometrial fragments, vaginal secretions and other mixtures. Therefore, the identification of blood and non-blood samples can be achieved through red blood cell-specific miRNA, and the identification of peripheral blood and menstrual blood samples can be achieved through specific miRNA of non-blood components in menstrual blood such as endometrium.

[0003] As intracellular regulatory molecules, miRNA participates in the regulation of gene expression and has individual differences. In 2014, Hanson et al. used logistic regression analysis to establish a menstrual blood identification model, which can accurately identify menstrual blood from four body fluids: peripheral blood, semen, saliva, and vaginal secretions, but lacks an identification scheme for peripheral blood. In 2016, Sauer et al. constructed two discriminant functions to achieve the identification of saliva from vaginal secretions and menstrual blood from peripheral blood, but failed to achieve the identification of menstrual blood and peripheral blood from non-blood samples. Previous studies have confirmed that the discriminant analysis method is suitable for the study of forensic body fluid identification based on miRNA markers, but none of them have achieved high-accuracy identification of forensic peripheral blood and menstrual blood. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to use the expression relationship of miRNA based on RNU6b as an internal reference gene to distinguish menstrual blood from peripheral blood. The present invention screened out three candidate miRNA molecules (miR-451a, miR-144-5p, miR-203-3p), used RNU6b with the highest stability as an internal reference gene, and established a method for efficiently and accurately distinguishing menstrual blood from peripheral blood based on miRNA.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The invention discloses a product for distinguishing menstrual blood from peripheral blood, characterized in that the product comprises a reagent for detecting the expression amount of miRNA in the body fluid to be tested; the miRNA is one or a combination of miR-451a, miR-144-5p or miR-203-3p.

[0007] Furthermore, the reagent for detecting mRNA levels includes primers for detecting miRNA, namely miR-451a, miR-144-5p or miR-203-3p, and the nucleotide sequences of the primers are shown in SEQ ID NO.1~3.

[0008] Furthermore, the product also includes the RNU6b internal reference gene.

[0009] Furthermore, the primer sequence of the RNU6b internal reference gene is shown in SEQ ID NO.4~5.

[0010] The present invention also discloses a primer probe set for distinguishing menstrual blood from peripheral blood, characterized in that the nucleotide sequences of the specific primers are shown in SEQ ID NO.1-3.

[0011] The present invention also discloses the use of the primer probe group for distinguishing menstrual blood from peripheral blood in preparing a reagent or a kit for distinguishing menstrual blood from peripheral blood.

[0012] Furthermore, an RNU6b internal reference gene is also included.

[0013] Furthermore, the primer sequence of the RNU6b internal reference gene is shown in SEQ ID NO.4~5.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0015] Based on high-throughput sequencing technology, the present invention screened out three miRNAs (miR-144-5p, miR-203-3p, miR-451a) with highly differential expression from vaginal blood and peripheral blood for the first time, and further verified the differences in screening indicators in three other body fluids (semen, saliva, and vaginal secretions). Ultimately, an efficient and accurate identification method system was established, which may become a powerful tool for determining the tissue source of body fluid stains in forensic evidence science, and provide more scientific and reasonable auxiliary evidence for the characterization and detection of cases.

[0016] 1) This project has screened out three miRNAs that are differentially expressed in blood and non-blood samples and in peripheral blood and menstrual blood samples based on the BGISEQ-500 sequencing platform for miRNA high-throughput sequencing technology.

[0017] 2) NormFinder software has been used to evaluate the stability of six reference genes.

[0018] 3) Real-time fluorescence quantitative PCR technology was used to detect the expression levels of three miRNAs in three types of body fluids.

[0019] The present invention uses six reference genes (RNU6b, U6, miR-103a-3p, miR-191-5p, miR-92a-3p, miR-484) to evaluate and compare the results, and finally selects RNU6b with the highest stability as the reference gene. At the same time, RNA sequencing and real-time fluorescence quantitative PCR technology are performed on female menstrual blood and peripheral blood samples to verify that miR-451a, which is differentially expressed in blood and non-blood samples, and two miRNAs (miR-203-3p, miR-144-5p) that are differentially expressed in peripheral blood and menstrual blood are selected as specific molecular markers for preliminary research work. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the technical route of the present invention.

[0021] Figure 2The bar graph shows the results of qPCR detection of six reference genes in five body fluids. PB: peripheral blood; SA: saliva; MB: menstrual blood; SE: semen; VA: vaginal fluid.

[0022] Figure 3 The results of the NormFinder software stability assessment of six internal reference genes. The six internal reference genes in the figure are ranked from large to small according to their stability values ​​(left). The smaller the stability value, the better the stability of the internal reference gene.

[0023] Figure 4 PCR expression levels of three miRNA genes in three types of body fluids.

[0024] Figure 5 The bar graph shows the average ΔCt values ​​of three miRNAs in different body fluid samples. Horizontal axis: pb: peripheral blood; sa: saliva; mb: menstrual blood; se: semen; va: vaginal fluid. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0026] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0027] 1. Research plan

[0028] 1 Materials.

[0029] Sample sources: 200 body fluid samples were collected from unrelated volunteers aged 20-25 years old in northern China, including 40 peripheral blood samples; 40 menstrual blood samples; 40 saliva samples; 40 semen samples; 40 vaginal secretions. In the pilot experiment, we first randomly selected 25 samples (5 samples of each body fluid) for the evaluation of internal reference genes.

[0030] Collection method: peripheral blood was collected by venipuncture into EDTA anticoagulation tubes, saliva was collected using sterile plastic tubes (fasting for 1 hour before sample collection), and semen was collected using sterile wide-mouth plastic cups (abstinence for 2 days before sample collection). The above liquid samples were directly stored at -80℃ for later use. Menstrual blood (4 days before the menstrual cycle) and vaginal secretions were collected by collecting tampons, dried at room temperature for one day, and then stored at -80℃ for later use.

[0031] 2Main instruments and reagents.

[0032] Table 1 Main instruments and equipment .

[0033] Table 2 Main reagents .

[0034] 3. Research methods.

[0035] 3.1 Selection of candidate miRNA molecular markers.

[0036] Using the BGISEQ-500 sequencing platform, high-throughput sequencing of miRNA was performed on menstrual blood and peripheral blood samples from four women, and three candidate miRNA molecules (miR-451a, miR-144-5p, miR-203-3p) were selected for this study. The selected candidate miRNA markers were verified by RT-qPCR for large samples. The expression levels of six reference genes (RNU6b, U6, miR-103a-3p, miR-191-5p, miR-92a-3p, miR-484) were evaluated by real-time fluorescence quantitative PCR, and RNU6b with the highest stability was finally selected as the reference gene. The information of each miRNA and reference gene is shown in Table 3 Table 3 mcroRNA markers and reference gene information .

[0037] 3.2 RNA extraction and quantification.

[0038] The total RNA was extracted from five body fluid samples using the miRNeasy Mini Kit (Baoxin Biotechnology, China) according to the instructions. 250 μL of peripheral blood, 500 μL of saliva, 300 μL of urine, and 2 cm of menstrual blood were extracted from each sample. 2 For sanitary napkins, take 1cm per vaginal secretion 2 Sanitary napkins. In addition, the mixed sample was added with 100 µL of peripheral blood sample and 1 cm of menstrual blood sample for each group. 2 The sanitary napkins were mixed into the same EP tube for extraction. The total RNA concentration and purity (A260 / 280) were determined using Nanodrop 2000c (Thermo Fisher Scientific, USA).

[0039] 3.3 Synthesis of cDNA.

[0040] This study used the stem-loop reverse transcription method, and the M-MLV Reverse Transcriptase (TaKaRa, Japan) reverse transcription kit was used for reverse transcription. A single reverse transcription system totaled 20 µL: 100 ng total RNA, 1 µL specific stem-loop reverse transcription primer, 4 µL 5× First-StrandBuffer (TaKaRa, Japan), 2 µL 0.1 mol / L DTT (TaKaRa, Japan), 0.5 µL dNTP Mixture (TaKaRa, Japan), 1 µL reverse transcriptase M-MLV (TaKaRa, Japan), 0.2 µL RecombinantRNasin® RNase Inhibitor (TaKaRa, Japan), and RNase-free water (TaKaRa, Japan) was added to the system to 20 µL. The reverse transcription program was 16°C for 30 min; 37°C for 30 min; 65°C for 5 min; and 4°C for incubation. A negative control for reverse transcription was set up (replacing reverse transcriptase with nuclease-free water). Specific stem-loop reverse transcription primer information Table 2.2 Table 4 Stem-loop reverse transcription primer information .

[0041] 3.4 qPCR.

[0042] This study used SYBR Green fluorescence quantitative PCR technology, and the reaction system was 10 µL: Power SYBR Green PCR Master Mix (2×) 5 µL; forward amplification primer (10 µmol / L) 0.25 µL; reverse amplification primer (10 µmol / L) 0.25 µL; nuclease-free water 4 µL; cDNA product 0.5 µL. Real-time PCR amplification program: 95°C 10 min; 95°C 15 s, 60°C 1 min, a total of 40 cycles; melting curve analysis was performed under the conditions of 95°C 15 s; 60°C 1 min. Real-time fluorescence quantitative PCR primer information is shown in Table 2.3. Real-time fluorescence quantitative PCR reactions were performed on a 7500 Real-Time PCR System (Applied Biosystems, USA). Data were collected using QuantStudioTM Real-Time PCR Software v1.3. Ct>35 was considered undetectable. Each sample was tested three times. Set up a qPCR negative control (replace sample with nuclease-free water as template).

[0043] Table 5 Real-time fluorescence quantitative PCR primer information .

[0044] 3.5 Statistical analysis

[0045] The relative expression data of each microRNA marker, ΔCt, were calculated: ΔCt=Ct(miRNA)-Ct(RNU6b). RNU6b was used as an internal reference gene to normalize the data. The relative expression data of 100 samples were randomly selected as training sets for data analysis and model construction. GraphPad Prism 7 software was used to draw the corresponding bar graphs. SPSS 22.0 software was used for normal distribution test (Kolmogorov-Smirnor test). For data that obeyed normal distribution and variance homogeneity, one-way analysis of variance was performed. If the data did not obey normal distribution or variance homogeneity, non-parametric tests (Mann-Whitney test or Kruskal-Wallis H test) were used for analysis. Bonferroni correction method was used for further pairwise comparisons between groups. NormFinder internal reference gene evaluation software was used to compare the stability of the six internal reference genes.

[0046] 2. Experimental results.

[0047] Screening and evaluation of internal reference genes.

[0048] Fluorescence quantitative PCR was used to detect the expression Ct values ​​of six reference genes in 25 samples of five body fluids, including peripheral blood, semen, menstrual blood, urine and vaginal secretions, and the corresponding bar graphs were drawn, see ( Figure 1 ),Depend on Figure 1 It can be seen that the six reference genes have different degrees of expression differences in the five body fluids.

[0049] The detection data of six reference genes (U6, RNU6b, miR-103a-3p, miR-191-5p, miR-92a, miR-484) were analyzed by NormFinder reference gene stability evaluation software. The results showed that the stability of the six reference genes was ranked from low to high: U6, miR-103a-3p, miR-92a, miR-191-5p, miR-484, RNU6b, see ( Figure 2 ).

[0050] Analysis of the expression of three miRNAs in five body fluids.

[0051] Fluorescence quantitative PCR was used to detect the expression of three miRNAs (miR-451a, miR-144-5P, miR-203-3P) in three types of body fluids: menstrual blood, peripheral blood, and non-blood, and the expression was expressed as Ct values ​​(see Figure 3), miR-451a was specifically expressed in non-blood samples, miR-203-3P was specifically expressed in peripheral blood samples, and miR-144-5P was specifically overexpressed in menstrual blood. The relative expression levels in the five body fluids were plotted as ΔCt bar graphs (see Figure 4 ), the three miRNAs are not completely specifically expressed in different body fluids, but there are significant differences in expression levels.

[0052] In summary, the present invention is the first to screen out three candidate miRNAs (miR-451a, miR-144-5P, miR-203-3P) with highly differential expression from vaginal blood and peripheral blood based on high-throughput sequencing technology, and further verify the differences in screening indicators in three other body fluids (semen, saliva, and vaginal secretions), establish Fisher discriminant analysis models for blood and non-blood samples and peripheral blood and peripheral blood samples, and establish an efficient and accurate method system for distinguishing menstrual blood from peripheral blood.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A product for distinguishing menstrual blood from peripheral blood, characterized in that: The product comprises a reagent for detecting the expression level of miRNA in a body fluid to be tested; the miRNA is one or a combination of miR-451a, miR-144-5p or miR-203-3p.

2. The product according to claim 1, characterized in that The reagent for detecting mRNA level includes primers for detecting miRNA, namely miR-451a, miR-144-5p or miR-203-3p, and the nucleotide sequences of the primers are shown in SEQ ID NO.1-3.

3. The product according to claim 1, characterized in that The product also includes the RNU6b internal reference gene.

4. The product according to claim 3, characterized in that The primer sequence of the RNU6b internal reference gene is shown in SEQ ID NO.4~5.

5. A primer probe set for distinguishing menstrual blood from peripheral blood, characterized in that: The nucleotide sequences including the specific primers are shown in SEQ ID NO. 1-3.

6. Use of the primer probe set for distinguishing menstrual blood from peripheral blood according to claim 5 in preparing a reagent or a kit for distinguishing menstrual blood from peripheral blood.

7. The use according to claim 6, characterized in that The RNU6b internal reference gene was also included.

8. The use according to claim 7, characterized in that The primer sequence of the RNU6b internal reference gene is shown in SEQ ID NO.4~5.

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