【 ^0001】 The present invention relates to a method and primer for a quantitative multiplex assay of microRNA for disease diagnosis, detection of pathogens, environmental contaminants, or the like.

BACKGROUND OF THE INVENTION

【 ^0002】 A microRNA (abbreviated miRNA) is a small non-coding RNA molecule, that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs function via binding to target microRNAs to regulate their expression.

【 ^0003】 Naturally occurring miRNAs are 19 to 25-nucleotide (nt) transcripts cleaved from 70 to 100 nt hairpin primary precursors, which are encoded in the genomes of invertebrates, Vertebrates and plants. Although, the biological functions of miRNAs remain to be fully understood, for the most part, these non-coding RNAs seem to regulate protein expression by either causing degradation or translation inhibition of the corresponding coding mRNA.

【 ^0004】 Quantitative assay of miRNAs has faced great difficulties and challenges due to the following reasons: (1) mature miRNAs are very small RNAs without poly(A)tail; (2) many miRNAs are highly conserved in sequence (in some cases there is only one-nucleotide difference); and (3) miRNAs generally express in a relatively low level. Nevertheless, scientists have developed several methods for miRNA assay, which can generally be classified into two categories: next generation sequencing (NGS) and polymerase chain reaction (PCR) -based methods.

【 ^0005】 The main purpose of the next generation sequencing is to explore unknown miRNAs, and to know the types of all miRNAs in the cell, however it is quite time-consuming and laborious in operation, making it unsuitable for high-throughput analysis. Furthermore, the result is presented as reads per million (RPM), which only the proportion of specific RNA in all RNAs can be obtained from, however, the RNA of interest cannot be accurately quantified.

【 ^0006】 Quantitative polymerase reaction (qPCR) is the currently the most common means to analyze miRNA expression. At present, there are several methods for miRNA expression analysis, including the use of SYBR Green or TaqMan probe to detect cDNA amplification.

【 ^0007】 SYBR Green binds to all double-stranded DNA, but it has no specificity, and the results are expressed as Ct values, which do not reflect the amount of cDNA.

【 ^0008】 The TaqMan method is not interfered by non-specific products and relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection. At present, qPCR machines can only measure up to eight specific products in one reaction well depending on the type of laser beam and fluorescent substance.

【 ^0009】 The present invention is in view of the technical insufficiency of microRNAs, and it is currently impossible to accurately quantify microRNAs by using NGS or qPCR techniques. Due to the lack of quantitative techniques for microRNAs, it is currently impossible to accurately quantify microRNAs using NGS or qPCR techniques. Therefore, the present invention utilizes a specific primer set to accurately amplify a specific microRNA, and the amount of microRNA can be quantified by a laser-stimulated stimulus by a probe on the microbead. SUMMARY OF THE INVENTION ^【0010】 Accordingly, the present invention is directed to a method and primer for a miRNA assay and application of the same which can overcome one or more disadvantages described above.

【 ^0011】 The present invention provides A method for determining the qualitative or quantitative status of a putative miRNAs in a sample, comprising the steps of: RNA Polyadenylation in which, the putative miRNA are polyadenylated with poly(A) polymerase to obtain an RNA-PolyA; first strand cDNA synthesis in which, using the RNA-PolyA as a template, the putative miRNA is then reverse transcribed into a complement cDNA using a JI primer comprising SEQ ID NO.01; PCR reaction in which, using the first strand cDNA as a template, a PCR reaction is performed with a specific forward primer designed to specifically hybridize to the first strand cDNA, and a universal reverse primer comprising SEQ ID NO.01, to give a PCR product; wherein the universal reverse primer further comprises a tag at the 5’ terminus; conjugation of beads with oligonucleotide probe in which, a oligonucleotide designed to specifically hybridized to the complementary strand of the first strand cDNA is coupled to a bead comprising one or more functional groups on its surface and a first detectable marker in its interior, to give a specific oligonucleotide probe; hybridizing the PCR product and the specific oligonucleotide probe to give a hybridization product; introducing a reporter to the hybridization product to give a hybridization mixture; wherein the reporter is capable of binding to the tag and has a second detectable marker. a qualitative or quantitative analysis is then performed on the hybridization mixture to identify the status of the putative miRNA in the sample. 【 ^0012】 In the method for determining the qualitative or quantitative status of a putative miRNAs in a sample, the tag is a biotin

【 ^0013】 In the method for determining the qualitative or quantitative status of a putative miRNAs in a sample, the functional groups are selected from the group consisting of amines, thiols, carboxylic acids, hydrazine, halides, alcohols, and aldehydes.

【 ^0014】 In the method for determining the qualitative or quantitative status of a putative miRNAs in a sample, the first detectable marker is a magnetic material. 【 ^0015】 In the method for determining the qualitative or quantitative status of a putative miRNAs in a sample, the reporter is a streptavidin coupled to the second detectable marker.

【 ^0016】 In the method for determining the qualitative or quantitative status of a putative miRNAs in a sample the second detectable marker is a fluorophore; wherein the fluorophore is PE (R-phycoerythrin).

【 ^0017】 In the method for determining the qualitative or quantitative status of a putative miRNAs in a sample, the qualitative or quantitative analysis comprising: flowing the hybridization mixture into a detection region; detecting the first detectable marker signals from the bead and the second detectable marker from the reporter; and determining status of the complementary strand of the first strand cDNA, wherein coincident detection of signals from the bead and the reporter indicates the complementary strand of the first strand cDNA is not fragmented, and wherein detecting a signal from only one of the bead or the reporter indicates the complementary strand of the first strand cDNA is fragmented.

【 ^0018】 the qualitative or quantitative analysis, the first detectable marker is a magnetic material.

【 ^0019】 the qualitative or quantitative analysis, the second detectable marker is a fluorophore; wherein the fluorophore is PE (R-phycoerythrin) BREIF DESCRIPTION OF THE DRAWINGS ^【0020】 FIG.1 depicts the schematic diagram of Quantitative multiplex PCR assay.

【 ^0021】 FIG.2 depicts the primer set (miR-375 forward and reverse primer). 【 ^0022】 FIG.3 depicts the MiR-375 probe. 【 ^0023】 FIG.4 depicts the synthetic sequence of MiR-375.

【 ^0024】 FIG.5 depicts the standard curve using microbead.

【 ^0025】 FIG.6 depicts the quantitative analysis of microbeads of PCR products under different temperature conditions.

【 ^0026】 FIG.7 depicts the quantitative analysis of microbeads of PCR products with different cycle numbers.

【 ^0027】 FIG.8 depicts the quantitative analysis of microbeads of PCR products with different concentrations of hsa-miR-375. DETAILED DESCRIPTION OF THE INVENTION 【 ^0028】 The present invention is in view of the technical insufficiency of microRNAs, and it is currently impossible to accurately quantify microRNAs by using NGS or qPCR techniques. Due to the lack of quantitative techniques for microRNAs, it is currently impossible to accurately quantify microRNAs using NGS or qPCR techniques. Therefore, the present invention utilizes a specific primer set to accurately amplify a specific microRNA, and the amount of microRNA can be quantified by a laser-stimulated stimulus by a probe on the microbead.

【 ^0029】 Example 1: Sample amplification

【 ^0030】 The poly(A) polymerase is used to ligate the poly A sequence to the microRNA (hsa-miR-375) at the 3’ end, and reverse transcription is performed into the micDNA using the JI sequence (Fig. 1) of the invention. The poly (A) tailing and reverse transcription is performed by using abm® miRNA cDNA synthesis kit with poly(A) polymerase tailing. The steps of poly (A) tailing and reverse transcription follows the manufacturer’s instruction. The hsa-miR-375 minic is used as RNA template. The syn-hsa-miR-375 is diluted to 4 µM and 2 µL of diluted miRNA is subjected to do poly (A) tailing and reverse transcription.

【 ^0031】 After the reverse transcription of hsa-miR-375 into cDNA, the polymerase chain reaction (PCR reaction) is used to amplify the hsa-miR-375 cDNA using a primer set: miR-375 forward and reverse primer. The PCR reaction is performed by using New England BioLab® Q5® high-fidelity 2X master mix kit. The PCR steps are followed by the manufacturer’s instruction. The 2 µL of cDNA is subjected to PCR reaction and cycle number is 35 cycles. The length of product is 43bp.

【 ^0032】 Example 2: beads conjugated with MiR-375 probe

【 ^0033】 A sequence is designed as a probe for the human micro RNA 375 gene, and amide modification is performed at the 5' end. Appropriate amount of well-mixed beads are mixed with 0.1M 2-(N-morpholono) ethanesulfonic acid (MES) buffer and 0.2nmol of MiR-375 probe (Fig. 3). The mixed beads are added 10mg/ml of freshly prepared 1-ethyl-3-3(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution and stood at room temperature for 30 minutes, repeated EDC step. Subsequently, the beads are followed by adding 0.5ml of 0.02% Tween 20 mixing well and centrifuged at 14000rpm for 3 minutes, remove the supernatant. Then added 0.5ml of 0.1% SDS solution, mixed well, centrifuged at 14000rpm for 3 minutes, removed the supernatant. Finally, the conjugated beads are resuspensed in appropriate amount of Tris-EDTA.

【 ^0034】 Example 3: Establishment of a standard curve for MicroRNA 375 【 ^0035】 The microRNA375 oligonucleotide, which is artificially biotinylated on the 5-terminal end, is used as a standard for the analysis platform (Fig. 4). The hybridization reaction is carried out on a 96 well PCR plate, and each well is prepared with 2500 magnetic beads and 5 µl of PCR product, and uniformly mixed, and the total volume of each well is 50 µl. The hybridization reaction is carried out in a PCR machine, and the hybridization conditions are as follows: reaction at 95 ° C for 5 minutes, reaction at 71.3 ° C for 10 minutes, and reaction on ice for 15 minutes, and then transfer the PCR well plate sample to the 96-well analysis black disk, and then use the Magnetic plate. The supernatant is removed, and 75 µl/well SA-PE is added to react at room temperature for 30 minutes. The Magpix instrument is used to detect the median Fluorescent Intensity (MFI) of its magnetic beads (Fig.5).

【 ^0036】 Example 4: The quantitative analysis of microbeads of PCR products 【 ^0037】 Annealing temperature: The fluorescence value of the PCR products is analyzed by microbeads. The 2 µL of 4µM standard syn-hsa-miR-375 is subjected to reverse transcription. The 2 µL of cDNA is amplified at different annealing temperatures (Fig.6). The optimum temperature is 55~58 °C.

【 ^0038】 PCR amplification cycle number: The fluorescence value of the PCR products is obtained by microbead analysis. The 2 µL of 4µM standard syn-hsa-miR-375 is subjected to reverse transcription. The 2 µL of cDNA is amplified at different cycle numbers. (Fig.7). The optimal number of cycles is 35.

【 ^0039】 Different concentrations of hsa-miR-375 minic: The 2µL of different concentrations standard product syn-hsa-miR-375 (1nM, 100pM, 10pM, 1pM) is subjected to reverse transcription and amplified at 35cycle numbers. The resulting beads are analyzed for fluorescence values (Fig.8).