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MiRNA-21 [micro-RNA (ribonucleic acid)-21] detection reagent kit on basis of digital PCR (polymerase chain reaction) platforms and application of miRNA-21 detection reagent kit

A detection kit, miRNA-21 technology, applied in the field of miRNA-21 detection kits, to achieve high sensitivity and accuracy, stable and reliable detection results, and reduce the pain of patients

Inactive Publication Date: 2017-03-22
成都仕康美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no convenient and quick kit for detecting miRNA-21 based on a digital PCR platform.

Method used

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  • MiRNA-21 [micro-RNA (ribonucleic acid)-21] detection reagent kit on basis of digital PCR (polymerase chain reaction) platforms and application of miRNA-21 detection reagent kit
  • MiRNA-21 [micro-RNA (ribonucleic acid)-21] detection reagent kit on basis of digital PCR (polymerase chain reaction) platforms and application of miRNA-21 detection reagent kit
  • MiRNA-21 [micro-RNA (ribonucleic acid)-21] detection reagent kit on basis of digital PCR (polymerase chain reaction) platforms and application of miRNA-21 detection reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: A detection kit for miRNA-21 based on a digital PCR platform

[0022] The kit consists of 9ul reverse transcription buffer, 2ul reverse transcriptase, 17ul PCR buffer, 20ul ultrapure water, 2ul positive control, 10ul quality control, 9ul quality control reverse transcription buffer solution and 17ul of quality control product PCR buffer;

[0023] Wherein, the reverse transcription buffer includes 100mM dNTPs (with dTTP) 0.15ul, 10×RT Buffer 1.5ul, 10mg / ml RNase inhibitor 0.2ul, 10uM reverse transcription primer 1ul and ultrapure water 6.15ul; Transcriptase is MultiScribe from ABI Company TM Reverse Transcriptase, 50U / μL; the nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO.1, specifically:

[0024] 5'-GTCGTATCCAGTGCGAGCCGTTGAGACCGCAAGTGCACTGGATACGACTCAACATC-3';

[0025] The PCR buffer includes 2×PCR Buffer 10ul (ddPCR from Bio-rad Company) TM EvaGreenSupermix), detection primer and ultrapure water 6ul, described detection prim...

Embodiment 2

[0030] Example 2: Detection of the expression level of miRNA-21 in healthy human plasma

[0031] 1. Sample collection and processing

[0032] Use Streck non-invasive blood collection tubes to collect 5ml of blood from healthy people. Transfer the blood to a new 15ml centrifuge tube, centrifuge at 2000g for 10min at 4°C, and draw the upper layer of plasma into a new 15ml centrifuge tube. Then centrifuge the plasma at 4000g for 20min at 4°C, and draw the upper layer of plasma into a new 15ml centrifuge tube. Draw 200ul of the upper layer of plasma and use QIAGEN's miRNeasySerum / Plasma Kit to extract total RNA according to the operating instructions. Note that during the extraction process, after the sample is treated with QIAzolLysis Reagent, add 10ul of the quality control product provided by the kit of the present invention, and finally extract 40ul of Total RNA samples.

[0033]Among them, the method of using the quality control product is: when using the kit to extract th...

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Abstract

The invention discloses an miRNA-21 [micro-RNA (ribonucleic acid)-21] detection reagent kit on basis of digital PCR (polymerase chain reaction) platforms and application of the miRNA-21 detection reagent kit. The miRNA-21 detection reagent kit comprises miRNA-21 detection reverse transcription primers, detection primers, quality control reverse transcription primers and detection quality control primers. The miRNA-21 detection reagent kit and the application have the advantages that components of reverse transcription buffer solution and PCR buffer solution are optimized, and accordingly long-term stabile storage can be facilitated, sample injection workload can be simplified and minimized, accordingly, possible faults or errors during sample injection can be reduced, and stable and reliable detection results can be obtained advantageously; human serum or plasma is selectively used as samples for the reagent kit, accordingly, the miRNA-21 detection reagent kit is convenient to store, miRNA-21 can be noninvasively detected, and suffering of patients can be relieved; the miRNA-21 detection reagent kit is based on the digital PCR platforms, and accordingly the sensitivity and the accuracy of the miRNA-21 detection reagent kit are superior to the sensitivity and the accuracy of other existing technological platforms when used for detecting trace nucleic acid in the serum or plasma.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit for miRNA-21 based on a digital PCR platform and an application thereof. Background technique [0002] MicroRNA (miRNA) is a non-coding small RNA molecule of about 22 nucleotides widely present in eukaryotes, which is crucial to cell development, differentiation, proliferation and apoptosis, and regulates and affects the entire life activity the process of. Studies in recent years have shown that miRNA can be used as a new generation of tumor markers, and has important application prospects and significance in cancer diagnosis and prognosis. [0003] A large number of studies have confirmed that miRNA-21-5p (miRNA-21) can be used as a tumor marker in breast cancer, liver cancer, lung cancer, colon cancer, gastric cancer, prostate cancer, pancreatic cancer, glioma, etc. Upregulated expression in cancer tissues. And in the serum or plasma samples of cance...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2563/159C12Q2525/207C12Q2527/125C12Q2545/113
Inventor 魏亮卢俊龚雪
Owner 成都仕康美生物科技有限公司
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