阅读说明：本技术 一种循环miR-1290的检测方法 (Detection method of circulating miR-1290 ) 是由 蔡建庭 徐丽怡 朱永良 陈肖 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种循环miR-1290的检测方法,包含以下步骤：(1)从检测样本血浆或血清中提取循环miRNA；(2)茎环法逆转录：将步骤(1)中得到的miRNA进行特异性茎环法逆转录；(3)qPCR检测：以步骤(2)中所获得的cDNA为模板,进行qPCR检测。本发明成本相对较低,而灵敏度和特异性较好。可用于人和动物等相关的基础和应用研究工作。(The invention discloses a detection method of circulating miR-1290, which comprises the following steps: (1) extracting circulating miRNA from the detected sample plasma or serum; (2) reverse transcription by a stem-loop method: carrying out reverse transcription on the miRNA obtained in the step (1) by a specific stem-loop method; (3) and (3) qPCR detection: and (3) carrying out qPCR detection by using the cDNA obtained in the step (2) as a template. The invention has relatively low cost and good sensitivity and specificity. Can be used for relevant basic and application research works of human, animals and the like.)

1. A method for detecting circulating miR-1290, which is characterized by comprising the following steps:

(1) extracting circulating miRNA from the detected sample plasma or serum;

(2) reverse transcription by a stem-loop method: carrying out reverse transcription on the miRNA obtained in the step (1) by a specific stem-loop method;

(3) and (3) qPCR detection: and (3) carrying out qPCR detection by using the cDNA obtained in the step (2) as a template.

2. The method for detecting circulating miR-1290 according to claim 1, wherein in the step (1), the extraction of circulating miRNA is realized by a nucleic acid adsorption column.

3. The detection method of circulating miR-1290 according to claim 1, characterized in that in the step (2), the reverse transcription system comprises a reverse transcription buffer, a reverse transcriptase, a reverse transcription primer, nuclease-free pure water and a miRNA template; the reverse transcription primer contains a specific stem-loop structure for accurately distinguishing highly homologous miRNAs; the nucleotide sequence of the reverse transcription primer aiming at the miR-1290 is shown as SEQ ID No. 1; the reaction conditions for reverse transcription were: bathing at 16 deg.C for 30min, at 37 deg.C for 30min, heating at 85 deg.C for 5min to inactivate enzyme, and storing at 4 deg.C.

4. The method for detecting circulating miR-1290 according to claim 3, wherein the total volume of a reverse transcription system is 20 μ L, wherein the reverse transcription buffer solution is 10 μ L, the reverse transcriptase is 1.5 μ L, the reverse transcription primer is 1 μ L, and the miRNA template diluted by nuclease-free pure water is 7.5 μ L.

5. The method for detecting circulating miR-1290 according to claim 1, wherein in the step (3), the reaction system for qPCR detection comprises: the kit comprises a Taqman premix, a specific forward primer, a universal reverse primer, a Taqman probe, nuclease-free pure water and a cDNA template; the nucleotide sequences of the specific forward primer, the Taqman probe and the universal reverse primer aiming at the miR-1290 are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 10; the reaction conditions for the qPCR assay were: pre-denaturation at 95 ℃ for 2.5min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, 40 cycles, and finally extension at 72 ℃ for 10 min.

6. The detection method of the circulating miR-1290 according to claim 5, characterized in that the total volume of the reaction system for qPCR detection is 10 μ L, wherein 5 μ L of Taqman premix, 0.2 μ L specific forward primer (10mM), 0.2 μ L, Taqman probe (10mM) universal reverse primer (10mM) and 4.2 μ L cDNA template diluted by nuclease-free pure water are used.

7. The method for detecting circulating miR-1290 according to claim 1, wherein in the step (1), an internal reference system is added, and the internal reference system comprises: internal references miR-16-5p and cel-miR-39 as well as corresponding reverse transcription primers, qPCR forward primers and Taqman probes,

the nucleotide sequence of the reverse transcription primer of miR-16-5p is shown as SEQ ID No.4,

the nucleotide sequence of the qPCR forward primer of miR-16-5p is shown in SEQ ID No.5,

the nucleotide sequence of the Taqman probe of miR-16-5p is shown in SEQ ID No.6,

the nucleotide sequence of the reverse transcription primer of cel-miR-39 is shown in SEQ ID No.7,

the nucleotide sequence of the qPCR forward primer of cel-miR-39 is shown in SEQ ID No.8,

the nucleotide sequence of the Taqman probe of cel-miR-39 is shown in SEQ ID No. 9;

the nucleotide sequence of miR-16-5p is shown in SEQ ID No. 12;

the nucleotide sequence of cel-miR-39 is shown in SEQ ID No. 13.

8. The method for detecting the circulating miR-1290 according to claim 7, wherein the internal reference cel-miR-39 is a 2-microliter solution of internal reference cel-miR-39 added in the sample extraction process, and the concentration is 0.2 uM.

9. A kit, which is prepared into a miR-1290 assay kit by the method for detecting circulating miR-1290 according to any one of claims 1 to 8.

10. The use of the circulating miR-1290 assay of a serum or plasma sample for the detection of miR-1290 expression level for basic research and clinical application according to the method for detecting miR-1290 of any one of claims 1-8.

Technical Field

The invention relates to the field of biomedicine, and in particular relates to a method for detecting circulating miR-1290.

Background

MicroRNA (miRNA) is a non-coding single-stranded small RNA with the length of about 19-23 nucleotides, widely exists in various organisms and is an important regulatory factor. mirnas regulate gene expression at the post-transcriptional level by complementarily pairing with target mrnas, degrading the target mrnas or inhibiting translation. The miRNA has high conservative property and strict space-time specificity, forms a complex regulation network with a target mRNA molecule thereof, and participates in various life activities including cell proliferation, apoptosis, cell differentiation, development, stress response and the like. mirnas play an important role in the development of tumors.

Research shows that miRNA can stably exist in various body fluids such as serum, plasma and the like, the miRNA is called circulating miRNA, is closely related to the occurrence and development of tumors, and can be used as an important marker for tumor screening, high-risk population prediction and treatment effect evaluation. Circulating mirnas are derived primarily from two pathways: passive leakage of tissue damage or apoptotic necrotic cells and active secretion of cell-derived microvesicles. Circulating mirnas exist independently of the cell, but have higher stability, probably due to annealing of RNA to DNA and thus resistance to both DNase and RNase degradation, or protection of RNA by inclusion in lipid or lipoprotein complexes, or by chemical modification or binding to proteins.

The existing miRNA detection methods mainly comprise Northern blot analysis, real-time fluorescence quantitative PCR (polymerase chain reaction), DNA microarray chips, second-generation sequencing and the like. The Northern blot analysis method is simple and easy to implement, but has low sensitivity, long time consumption, poor repeatability and larger required total RNA amount, and is not suitable for the detection of circulating miRNA. The microarray chip can perform high-throughput miRNA analysis, but still needs enough initial sample amount and has low sensitivity. The second-generation sequencing technology can also realize high-throughput detection of miRNA, but has higher detection cost and is not suitable for clinical analysis of large samples.

The qPCR detection is realized by adding a fluorescent group into a PCR reaction system and utilizing fluorescent signal accumulation to monitor the whole PCR process in real time, thereby realizing quantitative and qualitative analysis of the initial unknown content template. Compared to Northern blot and microarray analysis, qPCR requires a smaller amount of sample and significantly improves the detection range and sensitivity. However, since mirnas are very small, specially designed primers are required to achieve PCR detection. There are two main ways at present: poly (A) caudate and stem-loop methods. The poly (A) tailing method uses poly (A) polymerase to make a poly (A) sequence on the 3' end of miRNA, and then uses oligo (dT) sequence primer to do reverse transcription. Due to the universality of the reverse transcription primer, the detection cost is reduced, and the sensitivity and the specificity of detection are also reduced. The stem-loop method carries out reverse transcription on miRNA through a stem-loop primer, and because the 5 'end of the stem-loop primer contains a stem-loop structure and the 3' end of the stem-loop primer has 6 specific bases matched with the 3 'end of mature miRNA, short mature miRNA molecules can be expanded and a specific 3' primer site is added, so that the reverse transcription reaction can be carried out specifically.

Circulating miRNA detection is important content of liquid biopsy, and has important value for early diagnosis, curative effect evaluation and prognosis monitoring of tumors. Imaoka et al (Annals of Oncology 27: 1879-. The research of Ang Li et al (Clincancer Res; 19(13) J mu Ly 1,2013) finds that miR-1290 is remarkably increased in pancreatic cancer serum, the areas of the working curves (ROC) of the subjects of pancreatic cancer patients, chronic pancreatitis patients and pancreatic neuroendocrine tumor patients are respectively 0.96, 0.81 and 0.80, and circulating miR-1290 can be used as an early diagnosis marker of pancreatic cancer. The research of France sca Tavano et al (Sci Rep; 8 (1)) Nov 6,2018 shows that the combined detection of plasma miR-1290 and CA 19-9 has stronger diagnosis and identification efficiency than the separate detection of the two. Circulating miR-1290 is therefore a potential tumor marker.

Disclosure of Invention

The invention aims to provide a detection method of circulating miR-1290, which is a detection method for quantifying the circulating miR-1290 by combining stem-loop reverse transcription and taqman probe real-time fluorescence quantitative PCR.

The adopted technical scheme is as follows:

a method for detecting circulating miR-1290, comprising the following steps of:

(1) extracting circulating miRNA from the detected sample plasma or serum;

(2) reverse transcription by a stem-loop method: carrying out reverse transcription on the miRNA obtained in the step (1) by a specific stem-loop method;

(3) and (3) qPCR detection: and (3) carrying out qPCR detection by using the cDNA obtained in the step (2) as a template.

Further, in the step (1), the extraction of the circulating miRNA is performed by a nucleic acid adsorption column.

Further, in the step (2), the reverse transcription system comprises a reverse transcription buffer solution, a reverse transcriptase, a reverse transcription primer, nuclease-free pure water and a miRNA template; the reverse transcription primer contains a specific stem-loop structure for accurately distinguishing highly homologous miRNAs; the nucleotide sequence of the reverse transcription primer aiming at the miR-1290 is shown as SEQ ID No. 1; the reaction conditions for reverse transcription were: bathing at 16 deg.C for 30min, at 37 deg.C for 30min, heating at 85 deg.C for 5min to inactivate enzyme, and storing at 4 deg.C.

Further, the total volume of the reverse transcription system was 20. mu.L, wherein 10. mu.L of reverse transcription buffer, 1.5. mu.L of reverse transcriptase, 1. mu.L of reverse transcription primer, and 7.5. mu.L of miRNA template diluted with nuclease-free pure water.

Further, in the step (3), the reaction system for qPCR detection comprises: the kit comprises a Taqman premix, a specific forward primer, a universal reverse primer, a Taqman probe, nuclease-free pure water and a cDNA template; the nucleotide sequences of the specific forward primer, the Taqman probe and the universal reverse primer aiming at the miR-1290 are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 10; the reaction conditions for the qPCR assay were: pre-denaturation at 95 ℃ for 2.5min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, 40 cycles, and finally extension at 72 ℃ for 10 min.

Furthermore, the total volume of the reaction system for qPCR detection was 10. mu.L, wherein 5. mu.L of Taqman premix, 0.2. mu.L of specific forward primer (10mM), 0.2. mu. L, Taqman probe (10mM) for universal reverse primer (10mM), and 4.2. mu.L of cDNA template diluted in pure water without nuclease.

Further, in the step (1), an internal reference system is added, wherein the internal reference system comprises: internal references miR-16-5p and cel-miR-39 as well as corresponding reverse transcription primers, qPCR forward primers and Taqman probes,

the nucleotide sequence of the reverse transcription primer of miR-16-5p is shown as SEQ ID No.4,

the nucleotide sequence of the qPCR forward primer of miR-16-5p is shown in SEQ ID No.5,

the nucleotide sequence of the Taqman probe of miR-16-5p is shown in SEQ ID No.6,

the nucleotide sequence of the reverse transcription primer of cel-miR-39 is shown in SEQ ID No.7,

the nucleotide sequence of the qPCR forward primer of cel-miR-39 is shown in SEQ ID No.8,

the nucleotide sequence of the Taqman probe of cel-miR-39 is shown in SEQ ID No. 9;

the nucleotide sequence of miR-16-5p is shown in SEQ ID No. 12;

the nucleotide sequence of cel-miR-39 is shown in SEQ ID No. 13.

Further, the internal reference cel-miR-39 is an internal reference cel-miR-39 solution which is added in the sample extraction process and has the volume of 2 mu L, and the concentration is 0.2 uM.

A kit, which is prepared into a miR-1290 analysis kit by using the detection method of circulating miR-1290 in any scheme.

The application of the circulating miR-1290 detection method in any one of the schemes is to detect the miR-1290 expression level of a serum or plasma sample for basic research and clinical application.

The invention has the beneficial effects that:

the method is suitable for detecting the expression level of miR-1290 from the organism liquid sample with low miRNA abundance, and has relatively low cost, good sensitivity and specificity. According to the invention, the detection method can be prepared into a miR-1290 assay kit for in vitro detection and identification.

The detection method can be used for relevant basic and application research works of human, animals and the like.

The implementation of the invention has important social and economic benefits on the research, prevention and treatment and the like of the pathogenesis of malignant tumor seriously harming human health.

Drawings

FIG. 1 is an amplification curve of different concentrations of miR-1290 standard, with standard concentrations from left to right of 100nM, 10nM, 1nM, 100pM, 10pM and 1pM, respectively. In FIG. 1, RFU is relative fluorescence unit; cycles is the number of Cycles; amplification curve.

FIG. 2 is a standard curve for the miR-1290 standard.

FIG. 3 is a comparison of miR-1290 expression levels between serum and plasma samples from the same patient. Numbers N188, N189, N191, N192, N197, T45 and G112 are for different patients, respectively.

FIG. 4 is the relative expression levels of miR-1290 in different clinical samples.

Detailed Description

The invention is described in further detail below for the purpose of further understanding, but the scope of the invention is not limited thereto.

The detection method of the circulating miR-1290 mainly comprises the following three steps:

(1) extracting circulating miRNA from the detected sample plasma or serum;

(2) reverse transcription by a stem-loop method: carrying out reverse transcription on the miRNA obtained in the step (1) by a specific stem-loop method;

(3) and (3) qPCR detection: and (3) carrying out qPCR detection by using the cDNA obtained in the step (2) as a template.

In step (1):

1. the sample is serum or plasma; it may be preferred that the sample is serum.

2. Can select proper internal parameters

miR-16-5p is selected as an internal reference, and cel-miR-39 is exogenously added as another internal reference in the process of extracting nucleic acid. The sequence of miR-16-5p is shown in SEQ ID No.12, and the sequence of cel-miR-39 is shown in SEQ ID No. 13.

The internal reference cel-miR-39 solution is biosynthesized by Ruibo, the preparation concentration is 20um, and the use concentration is 10 nM.

In step (2):

3. design of specific Stem-Loop reverse transcription primers

Selecting a proper stem-loop sequence, adding a specific base paired with the 3 ' end of the mature miRNA sequence at the tail end, thereby forming a reverse transcription primer of which the 5 ' end contains a stem-loop structure, and the 3 ' end is provided with the specific base complementarily paired with the mature miRNA sequence, thereby expanding the short mature miRNA molecule and increasing the specific primer and probe binding site. The reverse transcription primers of each miRNA are respectively SEQ ID No.1, SEQ ID No.4 and SEQ ID No. 7.

The primers used were synthesized by Biotechnology engineering (Shanghai).

4. Design of specific qPCR primers and Taqman probes

According to the sequence and stem-loop sequence information of miR-1290, miR-16-5p and cel-miR-39, a specific upstream primer is designed, and the Tm values are ensured to be similar as much as possible (about 59 ℃). Meanwhile, a specific Taqman probe is designed, a modifying group at the 5 'end is 6-FAM, and a modifying group at the 3' end is TAMRA-N.

The probes used were synthesized by Biotechnology engineering (Shanghai).

5. Reverse transcription

The invention adopts miRNA first strand cDNA synthesis kit (NO: B532453) of biological engineering (Shanghai) company for reverse transcription. The reaction system comprises: mu.L of 2 XmiRNA L-RT Solution mix, 1.5. mu.L of miRNA-L-RT Enzyme mix, 1.0. mu.L of reverse transcription primer (10uM) and 7.5. mu.L of template, totaling 20. mu.L. The reaction conditions include 16 deg.C bath for 30min, 37 deg.C bath for 30min, 85 deg.C heating for 5min to inactivate enzyme, and 4 deg.C storage.

In step (3):

qPCR reaction and optimization of reaction conditions

The invention adopts the universal PCR downstream primer on the basis of the stem-loop reverse transcription, thereby reducing the experiment cost. Conventional Taqman premix (containing buffer, dNTPs Mix, regular Taq enzyme and MgCl) was used in the PCR process₂Solution) also increases the selectivity of the reagent. The technology of the invention has the advantages of strong specificity, high sensitivity, low cost, relatively simple operation and the like.

In conclusion, the invention adopts Taq PCR premix of Biotechnology engineering (Shanghai) company to carry out qPCR reaction. Optimizing a reaction system and reaction conditions to finally determine the optimal reaction system as follows: mu.L of Taq qPCR mix, 0.2. mu.L of specific forward primer (10uM), 0.2. mu.L of universal reverse primer (10uM), 0.4. mu.L of specific probe (10uM) and 4.2. mu.L of cDNA template, for a total of 10. mu.L. The reaction conditions are pre-denaturation at 95 ℃ for 2.5min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, 40 cycles, and finally extension at 72 ℃ for 10min, wherein the annealing temperature can be within the range of 50-60 ℃.

The invention is further illustrated by the following specific examples. Before describing the examples, the sample sources used for the various examples are described below in a unified manner:

the main sample sources of the invention are as follows:

peripheral blood samples were obtained from the second hospital affiliated with the Zhejiang university medical college. The serum/plasma collection procedure was: collecting blood in blood collecting tube containing coagulant, centrifuging at 3000rpm and 4 deg.C for 10min, and collecting supernatant (namely serum) for storage. Collecting blood in a blood collection tube containing an EDTA anticoagulant tube, centrifuging at 3000rpm and 4 ℃ for 10min, and taking supernatant in a 1.5ml centrifuge tube; centrifuging at 16000g and 4 deg.C for 10min, collecting supernatant (plasma), and storing. Serum/plasma samples were aliquoted and stored at-80 ℃.