Method for conducting multiplex quantitative detection on miRNA

A quantitative detection and multiplexing technology, applied in the field of fluorescence quantitative PCR, which can solve the problems of low sensitivity and low repeatability of detection results.

Active Publication Date: 2015-12-09
GENE TECH SHANGHAI COMPANY
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  • Description
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Problems solved by technology

High throughput is the biggest feature of this method, but its biggest shortcoming is its low sensitiv

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  • Method for conducting multiplex quantitative detection on miRNA
  • Method for conducting multiplex quantitative detection on miRNA
  • Method for conducting multiplex quantitative detection on miRNA

Examples

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Embodiment 1

[0052] Example 1, Quantitative detection of two miRNA examples

[0053] 1. Tested miRNA

[0054] In this example, miR-16 and miR-39 were quantitatively detected simultaneously by the method of the invention described above. miR-16 is a miRNA that is continuously expressed in the human body, and its expression level has a certain relationship with some diseases. miR-39 is an miRNA that is expressed in nematodes but not in humans, so it is often used as a quality control and added to samples. Simultaneous detection of miR-16 and miR-39 helps to standardize the detection of miR-16.

[0055] 2. Sequence design

[0056] (1) The sequences of miR-16 and miR-39 were obtained according to the database as follows:

[0057] miR-16: 5'-UAGCAGCACGUAAAUAUUGGCG-3' (SEQ ID NO: 1);

[0058] miR-39: 5'-UCACCGGGUGUAAAUCAGCUUG-3' (SEQ ID NO: 2)

[0059] (2) Reverse transcription primers, complementary primers and hydrolysis probes for miR-16 and miR-39 were designed as follows:

[0060] Re...

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Abstract

The invention relates to a method for conducting multiplex quantitative detection on miRNA. The method comprises the steps of reverse transcription, single-chain complementary extension, S1 nuclease digestion, solid phase adsorption extracting and fluorescent quantitative detection. According to the method for conducting the multiplex quantitative detection on the miRNA, on the basis of fluorescent quantitative PCR, the single-chain digestion function of S1 nuclease and the nucleic acid solid phase adsorption extraction technology are applied in a combined mode, and a breakthrough in the flux of miRNA quantitative detection is achieved.

Description

technical field [0001] The invention belongs to the technical field of fluorescent quantitative PCR, and relates to a fluorescent quantitative PCR method capable of quantitatively detecting two or more miRNAs at the same time. Background technique [0002] MicroRNA (miRNA) is a group of non-coding RNAs with a length of about 20-25 nucleotides that have the function of regulating gene expression. A large number of studies have shown that miRNA is involved in the occurrence and development of various diseases (such as tumors, autoimmune diseases, etc.), and the expression changes of miRNA can also be used to screen, diagnose or judge the prognosis of diseases. Establishing a stable and accurate quantitative detection method for miRNA is the prerequisite for its use as a biomarker and clinical diagnosis. Due to the double impact of individual variability and biological sample diversity, it is usually difficult to accurately predict the disease by a single miRNA expression chan...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2537/143C12Q2523/308C12Q2533/101
Inventor 梁旺王东李宾
Owner GENE TECH SHANGHAI COMPANY
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