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Helicase-dependent isothermal DNA amplification-based method for detecting microRNA

A constant temperature amplification and technical detection technology, applied in the field of biological analysis, can solve the problems such as the detection of microRNA that have not yet been seen, and achieve the effects of high specificity, high detection sensitivity, and reduction of background signals.

Active Publication Date: 2017-09-05
SHANDONG NORMAL UNIV
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Problems solved by technology

At present, there are few reports on HDA detection methods. The relevant research mainly uses HDA technology to detect some pathogenic bacteria, but there is no report using HDA method to detect microRNA.

Method used

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  • Helicase-dependent isothermal DNA amplification-based method for detecting microRNA
  • Helicase-dependent isothermal DNA amplification-based method for detecting microRNA
  • Helicase-dependent isothermal DNA amplification-based method for detecting microRNA

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Embodiment 1

[0058]Extraction of total intracellular RNA: Human cervical cancer cells (HeLa) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, Incubate at 37°C in an incubator containing 5% carbon dioxide. When the cells grow to the logarithmic growth phase, use the total RNA extraction kit (Cat. No. 74104) from KIAGEN, Germany, to extract and purify the total RNA in the cells. The extraction and purification operations are strictly in accordance with the instructions attached to the kit. Instruction manual. The obtained total RNA was used for quantitative detection of microRNA in cancer cells after the concentration was determined by UV-Vis spectrophotometer.

[0059] Exonuclease I (Exo I) Digestion: First prepare 19 μl of reaction buffer containing 1 mmol per liter of dithiothreitol (DTT), 1× Exonuclease I (Exo I) reaction Buffer, 20 units of RNase inhibitor, 10 mmol / L sodium chloride and 10 mmol / L Tris-HC...

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Abstract

The invention discloses a helicase-dependent isothermal DNA amplification-based method for detecting microRNA. The method comprises the following steps: (1) extracting total RNA in a sample; (2) adding a single-strand DNA probe and excessive exonuclease I into the extracted total RNA, incubating in a reacting buffer solution to realize specific combination of target microRNA and the single-strand DNA probe, and removing residual single-strand DNA probe by utilizing the excessive exonuclease I; and (3) adding upstream primer, downstream primer, single-strand binding protein and helicase to the reaction system, amplifying the target microRNA, and detecting expression of the target microRNA through a fluorescent signal. According to the method, the background is reduced by digesting the exonuclease I, and the signal is amplified by means of exonuclease assisted isothermal amplification (HDA) reaction, so that the microRNA can be quickly and sensitively detected.

Description

technical field [0001] The invention relates to the technical field of biological analysis, in particular to a method for detecting microRNA based on a DNA constant temperature amplification technology dependent on helicase. Background technique [0002] MicroRNA is a kind of endogenous non-coding single-stranded RNA discovered in a variety of eukaryotic cells and viruses in recent years. It is a short sequence of 21-25 nt in length. It is highly conserved in evolution and can be specific to target mRNA by Sexual base-complementary pairing can cause degradation of target mRNA or inhibit its translation, thereby regulating gene expression post-transcriptionally. Due to the conservation of microRNA sequences in different organisms, it is generally believed that the function of microRNA is involved in some basic processes of life, such as cell proliferation, cell death, stress response and fat metabolism during development. [0003] Therefore, microRNA is considered as a poten...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2521/513C12Q2522/101C12Q2521/319
Inventor 张春阳马飞刘萌
Owner SHANDONG NORMAL UNIV
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