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Digital PCR platform-based miRNA detection quality control kit and application method thereof

A technology for testing quality and kits, which is applied in the biological field, can solve the problems of greatly affecting the test results, and achieve the effects of avoiding PCR false positive amplification, simple operation, and not easy to degrade

Active Publication Date: 2017-02-15
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

Different from the detection of miRNA in human tissues or cells, U6 or RNU6B can be used as internal reference genes. At present, there is no clear and uniform serum or plasma miRNA internal reference, and the droplet digital PCR adopts the absolute quantitative method. The detection results It is displayed as the specific copy number concentration of the molecule to be detected in the sample. Therefore, miRNA extraction efficiency and miRNA reverse transcription efficiency have a great influence on the final detection result of digital PCR, but there is no convenient and quick miRNA for digital PCR platform Kits for Assay Quality Control

Method used

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  • Digital PCR platform-based miRNA detection quality control kit and application method thereof
  • Digital PCR platform-based miRNA detection quality control kit and application method thereof
  • Digital PCR platform-based miRNA detection quality control kit and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: a kind of miRNA detection quality control kit based on digital PCR platform, it comprises artificially synthesized miRNA, reverse transcription primer, detects upper primer, detects lower primer and DEPC water, and described artificially synthesized miRNA primer sequence is as SEQID As shown in No.1, the reverse transcription primer sequence is shown in SEQ ID No.2, the detection primer sequence is shown in SEQ ID No.3, and the detection primer sequence is shown in SEQ ID No.4 ;

[0028] The artificially synthesized miRNA concentration is 3×10 6 copies / μl, the volume is 8 μl, the concentration of the reverse transcription primer is 5 μM, and the volume is 1 μl; the concentration of the detection upper primer and the detection lower primer is 10 μM, each volume is 1 μl; the mass percentage concentration of the DEPC water It is 1%, and the volume is 1ml.

Embodiment 2

[0029] Embodiment 2: a kind of using method of miRNA detection quality control kit based on digital PCR platform, it comprises the following steps:

[0030] S1. Dilute the artificially synthesized miRNA to 1 / 10 times the original concentration with DEPC water;

[0031] S2. Extract the total miRNA of the biological body fluid as a sample, add lysate buffer to lyse the RNase in the sample, and add 4 μl of diluted artificially synthesized miRNA to the sample;

[0032] S3. Set the final dissolving volume of the total miRNA in the extracted sample to be 10-60 μl (volume A), and prepare a control sample. The specific steps are: draw 4 μl of diluted artificially synthesized miRNA and dissolve it in (A-4 μl) volume of DEPC water;

[0033] S4. While reverse-transcribing the target miRNA to be detected in the sample, reverse-transcribe the artificially synthesized miRNA in the sample and the control sample separately, and add 1 μl of reverse transcription primer to each 10 μL reaction s...

Embodiment 3

[0039] Example 3: Quality control in the process of detecting the expression level of miRNA-122 in healthy human plasma based on a digital PCR platform

[0040] 1. Sample collection and processing

[0041] Use Streck's Cell-Free DNA BCTTM tube 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. Pipette 200 μL of plasma into a new 1.5ml EP tube, and use QIAGEN's miRNeasy Serum / Plasma Kit to extract total RNA. Among them, after adding QIAzolLysis Reagent and standing for 5 minutes, 4 μL of artificially synthesized miRNA (hereinafter referred to as Skm-QCmiR) diluted by 10 times in this kit was added, and the subsequent experimental operation was carried out according to the operation manual, and the final disso...

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Abstract

The present invention discloses a digital PCR platform-based miRNA detection quality control kit, which comprises a synthesized miRNA, a reverse transcription primer, a detection upper primer, a detection lower primer and DEPC water. The invention also discloses an application method of the kit. The synthesized miRNA provided by the kit absolutely has no similar sequence in a human body, so that the false positive amplification of PCR is avoided. The digital PCR platform-based miRNA detection quality control kit can be used for the quality control of an exogenous internal reference miRNA used for digital PCR detection. Since the miRNA molecule itself is stable in structure and not prone to degradation, the long-term and stable preservation of the miRNA molecule is realized. In addition, by adopting the reverse transcription primer and the detection primers provided by the kit, stable and reliable detection results can be obtained during the detection of a digital PCR platform. During usage, the kit is simple in operation and convenient to use, thus being suitable for mass detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a miRNA detection quality control kit based on a digital PCR platform and a use method thereof. Background technique [0002] Droplet Digital PCR (Droplet Digital TM PCR) is called the third-generation PCR technology. Generally, the abundance of miRNA molecules in human body fluids such as plasma and urine is generally not high. Conventional PCR technology has large errors or is difficult to detect when detecting such miRNA molecules. Due to the major innovation and breakthrough of digital PCR in micro-detection technology, it has become the most recognized technical method in liquid biopsy. [0003] Based on the digital PCR platform, in the process of miRNA detection in human body fluids such as serum or plasma, three steps are required: extraction of total miRNA, reverse transcription of miRNA, and detection of miRNA. Different from the detection of miRNA in human tis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6883C12Q2600/166C12Q2600/178C12Q2525/207C12Q2545/107
Inventor 魏亮卢俊杨洪吉
Owner 成都仕康美生物科技有限公司
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