TWI689594B - Multiplex quantitative method for microrna - Google Patents

Abstract

The present invention relates to a technique for quantifying miRNA. In the polyermease chain reaction, the reverse primer is replaced with a biotinylated reverse primer, and a specific sequence on the microbead is used to bind to the the negative strand of the PCR product. The streptavidin-PE is conducted with the product and the Magpix instrument is used to detect the Median Fluorescent Intensity (MFI). The measurement of two or more miRNAs is distinguished by the numbering on the microbeads. Currently, the Magpix instrument can identify up to more than 10 specific products in a single reaction well.

Description

Method for quantifying microribonucleic acid by multiplexing

The invention relates to a method for multiplexing and quantifying micro ribonucleic acid and the primers used therein. The micro ribonucleic acid can be applied to the fields of disease diagnosis, pathogen detection, environmental pollutant inspection and the like.

MicroRNA (microRNA, miRNA for short) is a small non-coding RNA molecule, which acts on RNA silencing and post-transcriptional regulation of gene expression. The microRNA with target function can be combined with the target to regulate its performance .

Naturally occurring miRNAs are transcripts with about 19 to 25 nucleotides (nt), which are cleaved from hairpin primary precursors encoded in the invertebrate, vertebrate, and plant genomes with 70 to 100 nt. Although the biological functions of miRNAs are still not fully understood, in most cases, these non-coding RNAs seem to regulate protein performance by causing degradation of the corresponding coding mRNA or inhibiting its translation.

Quantitative analysis of miRNAs faces great difficulties and challenges due to the following reasons, including: (1) mature miRNAs are small fragments of RNA without poly(A) tail; (2) many miRNAs have Medium to high sequence conservation (in some cases, there is only one nucleotide difference); (3) miRNA performance is relatively low. However, scientists have developed many miRNA assays Methods can be roughly divided into two categories: next generation sequencing (NGS) and measurement methods based on the use of polymerase chain reaction (PCR).

The main purpose of next-generation sequencing is to understand the unknown miRNAs and the types of all miRNAs in the cell, but the operation process is wasteful and laborious, so it is not suitable for high-throughput analysis. In addition, the results are all based on The reads per million (RPM) method is presented, so only a specific proportion of RNA can be obtained from all RNA. However, the target RNA cannot be accurately quantified.

Quantitative polymerase chain reaction (qPCR) is one of the most commonly used methods for analyzing miRNA performance. There are several miRNA performance analysis methods, including the use of SYBR Green or TaqMan probes to detect cDNA amplification.

SYBR Green will bind to all double-stranded DNA, but because it is not specific, and the results are expressed in Ct values, and cannot reflect the amount of cDNA.

The TaqMan method is not disturbed by non-specific products, and when hybridizing with complementary target sequences or detecting fluorophores, the 5'-3' exonuclease activity of Taq polymerase is required to cleave the double label probe needle. Currently, depending on the type of laser beam and fluorescent substance, the qPCR instrument can only measure up to about 8 specific products in one reaction well.

In view of the shortcomings of the current quantitative miRNA technology, such as the inability to accurately quantify miRNA using NGS or qPCR technology, the present invention uses a specific primer set to accurately amplify a specific miRNA, and can pass through the microbead (microbead) ) The probe on) is stimulated by laser light to quantify miRNA.

Therefore, the present invention relates to a method for miRNA determination, primers used and applications thereof, which can overcome one or more disadvantages of the prior art.

The present invention provides a method for qualitatively or quantitatively determining a hypothetical miRNA in a sample, which includes the following steps: RNA polyadenylation, and poly(A) polymerase polyadenylation of the hypothetical miRNA Acidify to form RNA-PolyA; the first strand of cDNA synthesis uses the RNA-PolyA as a template to reverse transcribe the putative miRNA using a JI primer containing SEQ ID NO.01 to synthesize a first strand of complementation DNA (complementary DNA, cDNA); PCR reaction, using the first strand of cDNA as a template, and using a forward primer designed to specifically hybridize with the first strand of cDNA (forward primer) and a SEQ ID NO. The 01 sequence of universal reverse primer (Universal reverse primer) polymerase chain reaction (PCR) to obtain a PCR reaction product, wherein the universal reverse primer may further include a tag located at the 5'end; magnetic beads and oligonucleosides Acid probe conjugation, an oligonucleotide designed to specifically hybridize to the complementary strand of the first strand of cDNA and a magnetic bead containing one or more functional groups on its surface and a first in the magnetic bead Detectable label junction to obtain a specific oligonucleotide probe; hybridize the PCR reaction product with the specific oligonucleotide probe to obtain a hybridized product; add a reporter (reporter) to the hybridized product ), a hybridization mixture is obtained, wherein the reporter can be combined with the tag to form a second detectable label; and the hybridization mixture is subjected to qualitative or quantitative analysis to identify the miRNA in the sample.

In the method for qualitatively or quantitatively determining miRNA in a sample, the tag is a biotin.

In the method for qualitatively or quantitatively determining miRNA in a sample, the functional group is selected from the group consisting of amine, thiol, carboxylic acid, hydrazine, and halide ( halide), alcohol and aldehyde.

In the method for qualitatively or quantitatively determining miRNA in a sample, the first detectable label is a magnetic substance.

In the method for qualitatively or quantitatively determining miRNA in a sample, the reporter is streptavidin conjugated to the second detectable label.

In the method for qualitatively or quantitatively determining miRNA in a sample, the second detectable label is a fluorophore, wherein the fluorophore is PE (R-phycoerythrin).

In the method for qualitatively or quantitatively determining miRNA in a sample, the qualitative or quantitative analysis includes: flowing the hybridization mixture into a detection area; detecting the first detectable label signal from the magnetic beads and from the The second detectable label of the reporter; determine the status of the complementary strand of the first strand of cDNA, wherein if the signal coupling from the magnetic bead to the reporter is detected, it means that the complementary strand of the first strand of cDNA is not Fragmentation, if only one of the signals from the magnetic beads and the reporter is detected, it means that the complementary strand of the first strand of cDNA is fragmented.

In the quantitative or qualitative analysis, the first detectable label is a magnetic substance.

In the quantitative or qualitative analysis, the second detectable label is a fluorophore, wherein the fluorophore is PE (R-phycoerythrin).

FIG. 1 is a schematic diagram showing PCR multiplexing quantification.

Figure 2 shows the primer set (forward primer, reverse primer of miR-375).

Figure 3 shows the MiR-375 probe.

Figure 4 shows the synthetic sequence of MiR-375.

Figure 5 shows a standard curve using micromagnetic beads.

Figure 6 shows the quantitative analysis of micromagnetic beads of PCR products under different temperature conditions.

Figure 7 shows the quantitative analysis of micromagnetic beads for PCR products obtained using different cycles.

Figure 8 shows the quantitative analysis of the micromagnetic beads of PCR products obtained using different concentrations of hsa-miR-375.

In view of the shortcomings of the current quantitative miRNA technology, such as the inability to accurately quantify miRNA using NGS or qPCR technology, the present invention uses a specific primer set to accurately amplify a specific miRNA, and can excite micromagnetic beads through laser On the probe, which in turn produces a fluorescent reaction to quantify miRNA.

Example one, sample increase

The poly(A) polymerase was used to attach the poly(A) sequence to the 3'end of microRNA (hsa-miR-375), and the reverse transcription of microDNA was performed using the JI sequence (see Figure 1). The polyadenylation tailing and reverse transcription use the abm ^® miRNA cDNA synthesis reagent set. All steps refer to the instruction manual. The general steps are to use the hsa-miR-375 as the RNA template and the synthesized hsa-miR -375 (syn-hsa-miR-375) was diluted to 4 μM, and 2 μL of diluted syn-hsa-miR-375 was taken for subsequent polyadenylation tailing and reverse transcription.

After reverse transcription of hsa-miR-375 to cDNA, a primer set (miR-375 forward primer and reverse primer) was used to perform PCR reaction to amplify the hsa-miR-375 cDNA. This PCR reaction was performed using New England BioLab ^® Q5 ^® high-fidelity 2X master mix reagent set. All steps refer to the instruction manual. The approximate steps are to perform PCR reaction with 2 μL of cDNA and the number of cycles is 35, and the final product The length is 43bp.

Example 2: Magnetic beads and MiR-375 probe

Design a sequence as a probe for detecting the human microRNA 375 gene and modify the amide at its 5'end. Mix the appropriate amount of magnetic beads with 0.1M MES buffer (2-(N-morpholono)ethanesulfonic acid), 0.2nmol of the MiR-375 probe (as shown in Figure 3), and then add to the mixed magnetic beads 10mg/ml freshly prepared EDC solution (1-ethyl-3-3(3-3-dimethylaminopropyl)carbodiimide hydrochloride), and after standing at room temperature for 30 minutes, repeat the EDC step again, and then add 0.5ml 0.02 % Tween 20 and mix well, and centrifuge at 14000 rpm for 3 minutes to remove the supernatant, and finally add an appropriate amount of Tris-EDTA to suspend the conjugated magnetic beads.

Example 3: Establish the standard curve of MicroRNA 375

It was biotinylated at the 5'end of microRNA 375 and used as a standard to serve as an analysis platform (see Figure 4). Hybridization reactions were performed on 96-well PCR well plates. Each well added 2500 magnetic beads and 5 μL The PCR products were then mixed well, and the total volume of each well was approximately 50 μL. The hybridization reaction is carried out in a PCR instrument, and various hybridization conditions are as follows: reaction at 95°C for 5 minutes; reaction at 71.3°C for 10 minutes; reaction on water 15 minutes; transfer the sample from the PCR well plate to a 96-well analysis black well plate, and then use a magnetic plate; remove the supernatant, and add 75 μL of SA-PE to each well, and react at room temperature for 30 minutes; finally, use The Magpix instrument detects the mean fluorescent intensity (MFI) on the magnetic beads (the results are shown in Figure 5).

Example 4: Quantitative analysis of magnetic beads in PCR products

Annealing temperature: Analyze the fluorescence value of PCR products through micromagnetic beads, reverse transcription 2μL of 4μM standard syn-hsa-miR-375, and increase 2μL of cDNA under different annealing temperature conditions (see Figure 6), The best temperature is 55-58℃.

PCR amplification cycle number: the fluorescence value of PCR products was analyzed by micromagnetic beads, 2 μL of 4 μM standard syn-hsa-miR-375 was reverse transcribed, and 2 μL of cDNA was amplified under different cycle number conditions (see Figure 7) ), the optimal number of cycles is 35.

Different concentrations of hsa-miR-375 minic: 2μL of different concentrations of standard syn-hsa-miR-375 (1nM, 100μM, 10μM, 1μM) were reverse transcribed and increased by 35 cycles, which was obtained by subsequent analysis The fluorescence value of the magnetic beads (see Figure 8).

<110> Biotechnology Development Center

<120> A multiplex method for quantifying tiny ribonucleic acid

<130> None

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<170> PatentIn version 3.5

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Claims (11)

A method for quantitatively or qualitatively determining the micro-ribonucleic acid (miRNA) in a sample, including the following steps: a. RNA polyadenylation, which uses poly(A) polymerase to perform polyadenylation of miRNA to obtain A RNA-PolyA; b. The first strand of cDNA synthesis, using the RNA-PolyA as a template, using a primer containing the nucleotide sequence listed in SEQ ID NO.01 to perform reverse transcription to synthesize a complementary DNA ( cDNA); c. PCR reaction, using the first strand of cDNA as a template, and using a forward primer designed to specifically hybridize with the first strand of cDNA (forward primer) and a containing as shown in SEQ ID NO.01 The universal reverse primer of the nucleotide sequence is subjected to a polymerase chain reaction (PCR) to obtain a PCR reaction product, wherein the universal reverse primer further includes a tag at the 5'end; d. magnetic beads Conjugated with an oligonucleotide probe, an oligonucleotide designed to specifically hybridize to the complementary strand of the first strand of cDNA and a surface containing one or more functional groups and an internal A detectable labeled magnetic bead is joined to obtain a specific oligonucleotide probe; e. the PCR reaction product is hybridized with the specific oligonucleotide probe to obtain a hybrid product; f. in the hybridization A reporter is added to the product to obtain a hybridization mixture, wherein the reporter can be combined with the tag to form a second detectable label; and g. Perform a qualitative or quantitative analysis on the hybridization mixture to identify the sample The degree of miRNA. The method of claim 1, wherein the tag is a biotin. The method according to claim 1, wherein the functional group is selected from the group consisting of amine, thiol, carboxylic acid, hydrazine, halide, alcohol ) And aldehyde (aldehyde). The method of claim 1, wherein the first detectable mark is a magnetic substance. The method according to claim 1, wherein the reporter is streptavidin. The method of claim 5, wherein the second detectable mark is a fluorophore. The method according to claim 6, wherein the fluorophore is PE (R-phycoerythrin). The method of claim 1, wherein the qualitative or quantitative analysis in step g includes: flowing the hybridization mixture into a detection area; detecting the first detectable label signal from the magnetic beads and from the reporter The second detectable label; and determine the status of the complementary strand of the first strand of cDNA. The method of claim 8, wherein the first detectable mark is a magnetic substance. The method of claim 8, wherein the second detectable mark is a fluorophore. The method according to claim 10, wherein the fluorophore is PE (R-phycoerythrin).

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