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Detection method of single-stranded micro ribonucleic acid

A microRNA and detection method technology, applied in the field of single-stranded microRNA detection, can solve problems such as difficulties and low expression levels, and achieve the effects of simple detection, good detection specificity, and saving detection samples

Inactive Publication Date: 2010-08-04
TIANGEN BIOTECH BEIJING
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Problems solved by technology

Mature miRNAs generally have only a few dozen bases, and there is usually only one or two bases difference between miRNAs of the same family, and most of them have relatively low expression levels in cells. These characteristics give traditional detection methods such as ribose Conventional methods such as nucleic acid hybridization and chip detection have brought great difficulties and challenges

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  • Detection method of single-stranded micro ribonucleic acid
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  • Detection method of single-stranded micro ribonucleic acid

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Embodiment Construction

[0011] The detection method of the single-stranded microRNA proposed by the present invention may further comprise the steps:

[0012] (1) Add the ribonucleic acid comprising single-stranded microribonucleic acid to the polyadenylate polymerase reaction system, and react at 37°C for 50-70 minutes to obtain a reaction solution, and the polyadenylate polymerase reaction The volume of the system is 20 microliters, the mass-to-volume ratio of ribonucleic acid added is 2 nanograms to 200 nanograms / microliter, and the composition of the polyadenylate polymerase reaction system is: polyadenylate polymerase is 2 units , 20 nanograms to 2 micrograms of ribonucleic acid, 100 mmol / L of tris hydrochloride with a pH value of 8.0, 200 mmol / L of sodium chloride, and 80 mmol / L of ethylenediaminetetraacetic acid and adenosine triphosphate at 50 mmol / L;

[0013] (2) Take out 2 microliters from the reaction solution in step (1), add it to the reverse transcription reaction system, and react at ...

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Abstract

The invention relates to a detection method of single-stranded micro ribonucleic acid (miRNA), belonging to the technical field of biological detection. Cells are used or RNA (containing miRNA) is organized, wherein a polyadenylic acid tail is added to the 3' terminal of miRNA by poly (a) polymerase, then reverse transcription primers are used for reverse transcription to obtain single-stranded deoxyribonucleic acid (ssDNA) complementary to the RNA, finally the miRNA related sequences and partial complementary sequences of the reverse transcription primers are respectively used as the upperstream and downstream primers for miRNA detection and a specific detection system (containing SYBR Green I fluorescent dye) is used for detecting miRNA. The method has the advantages of simpleness, convenience, rapidness and suitability for detection of a large number of samples. The method can be used for expression analysis, cloning research and clinical sample test of miRNA.

Description

technical field [0001] The invention relates to a method for detecting single-stranded microRNA, belonging to the technical field of biological detection. Background technique [0002] Single-stranded micro ribonucleic acid (hereinafter referred to as miRNA) is a kind of ribonucleic acid molecule with a length of 21-23 single nucleotides, which is encoded by the genome of higher eukaryotes. RISC degrades messenger RNA or hinders its translation. miRNAs are quite conserved in the evolution of species. miRNAs found in plants, animals and fungi are only expressed in specific tissues and developmental stages. The tissue specificity and timing of miRNAs determine the functional specificity of tissues and cells, indicating that miRNAs are important in cell growth. and play a variety of roles in the regulation of developmental processes. The miRNA sequence is conserved in different biological cells. The function of miRNA is to participate in some basic processes of life, such as ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 寇彤俞萍李晓晨孙克非
Owner TIANGEN BIOTECH BEIJING
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