Quantitative detection method for micro RNA (Ribose Nucleic Acid)

A quantitative detection method and quality technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of poor specificity and sensitivity of the three-step detection method, and achieve improved sensitivity and accuracy Effects of monitoring, enhancing sensitivity

Active Publication Date: 2012-09-19
天津旷博同生生物技术有限公司
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Problems solved by technology

However, the specificity and sensitivity of conventional three-step assays are relatively poor

Method used

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  • Quantitative detection method for micro RNA (Ribose Nucleic Acid)
  • Quantitative detection method for micro RNA (Ribose Nucleic Acid)
  • Quantitative detection method for micro RNA (Ribose Nucleic Acid)

Examples

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Embodiment 1

[0022] Example 1 Detection of Hsa-miR-103a content in serum

[0023] The sequence of Hsa-miR-103a is 5'-AGCAGCAUUGUACAGGGCUAUGA-3' (SEQ ID NO: 7).

[0024] 1. Extraction of total RNA from serum

[0025] Take 2ml of blood from each of two adults, centrifuge after coagulation, and finally take 1ml of the upper serum and place it in a 1.5ml RNase / DNase-free centrifuge tube.

[0026] Use the RNA extraction kit (Beijing Kuangbo Biotechnology Co., Ltd.) to extract total RNA from serum, and add 1 μl (20 nM) of the sequence 5'-CAACCTCCTAGAAAGA-3' (SEQ ID NO: 1) to each 250 μl of serum. 1 (synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) to monitor the quality of RNA extraction from serum. The concentration of extracted total RNA was determined using Thermo NanoDrop 2000c.

[0027] 2. Three-step method for quantitative detection of HSA-miR-103a in serum

[0028] (1) Add polyA tail:

[0029] i. Prepare the polyA-tailed reaction solution in an RNase-free PCR tube ...

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Abstract

The invention discloses a quantitative detection method for micro RNA (Ribose Nucleic Acid), and the method comprises the following steps of: (1) extracting total RNA of a sample; (2) treating the total RNA by poly A polymerase, and adding a section of poly A tail at a 3' end of the mi RNA; (3) carrying out inverse transcription for oligo (dT) inverse transcription primer with joint sequence at a5' end, and adding a section of joint at the obtained first-chain c DNA (Deoxyribonucleic Acid); and (4) enabling sequence specificity of the mi RNA to be tested and general reverse primer complementary with the joint sequence to carry out quantitative real-time PCR (Polymerase Chain Reaction) amplification, wherein the general reverse primer is mixture of two types of reverse primers with different length. By means of the method disclosed by the invention, quantitative detection and analysis for mi RNA expression can be achieved, and the specificity, sensitivity and repeatability of the existing detection method can be greatly improved.

Description

technical field [0001] The invention relates to the field of nucleic acid detection, in particular to a three-step quantitative detection method for microRNA. This method is mainly used in microRNA expression profile analysis, microRNA clinical diagnosis, microRNA research and detection, and microRNA-related drug research and screening. Background technique [0002] microRNA (miRNA) is a class of non-coding RNA with regulatory functions about 20-24 nucleotides (nt) in length. miRNA is mainly involved in the regulation of gene post-transcriptional level. The miRNA gene is first transcribed into a precursor transcript (primary transcripts miRNA, pri-miRNA) in the nucleus, which is cut to form a miRNA precursor (pre-miRNA) of about 60-70nt under the action of a ribonuclease Drosha; After reaching the cytoplasm, another ribonuclease, Dicer, cuts it into miRNA:miRNA double strands of about 22nt. This double-stranded miRNA is quickly guided into the RNA-induced silencing comple...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 李启靖
Owner 天津旷博同生生物技术有限公司
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