Method and primers for detecting mi ribonucleic acid (miRNA) and application of method

A quantitative detection, universal primer technology, applied in the field of biomedicine, can solve problems such as expensive, achieve the effect of low cost and simple operation

Active Publication Date: 2011-08-17
苟德明 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The stem-loop primer method uses primers with 6 bases at the 3' end that are complementary to the miRNA for reverse transcription of the miRNA. At the same time, the 5' end of the reverse transcription primer contains a stem-loop structure, which can enhance the heterogeneous duplex of miRNA and DNA.

Method used

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  • Method and primers for detecting mi ribonucleic acid (miRNA) and application of method
  • Method and primers for detecting mi ribonucleic acid (miRNA) and application of method
  • Method and primers for detecting mi ribonucleic acid (miRNA) and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Quantitative detection of hsa-miR-21miRNA in female cervical cancer Hela cells

[0056] (1) Materials

[0057] 1. Materials:

[0058] Female cervical cancer Hela cells cultured in vitro.

[0059] 2. Reagents:

[0060] RNAiso plus (TaKaRa), PolyA Polymerase (EPICENTRE), PrimeScript RTreagent Kit (TaKaRa), SYBR Premix Ex Taq (Peffect Real Time) (TaKaRa), Premix Ex Taq (Perfect Real Time) (TaKaRa).

[0061] 3. Primers and probes:

[0062]

[0063] 4. Instrument:

[0064] GeneQuant pro RNA / DNA quantitative analyzer, ABI PCR instrument (2720), ABIreal-time PCR instrument (PRISM 7300).

[0065] (2) Method

[0066] 1. RNA extraction

[0067] RNA was extracted from Hela cells cultured in vitro with RNAiso plus reagent. The OD260 / 280 and RNA concentration of the extracted Hela cell total RNA were measured by a spectrophotometer, and the integrity of the RNA was analyzed by agarose gel electrophoresis.

[0068] 2. RNA tailing reaction

[0069] The total RNA...

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Abstract

The invention discloses a method and primers for detecting mi ribonucleic acid (miRNA) and application of the method. The method comprises the following steps of: performing reverse transcription on the miRNA in a sample by using a reverse transcription primer formed by specific basic groups and Oligo (dT); and performing real-time polymerase chain reaction (PCR) quantitative detection on the miRNA by using a specific forward primer, a general reverse primer and a general probe. The invention has the characteristics that: the sensitivity and the specificity of the method are obviously higher than those of the conventional method; high-throughput analysis can be performed; and the method is simply and quickly operated, is low in cost, and can be widely used for early diagnosis and prediction of critical diseases such as tumors and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method and primers for detecting miRNA and its application in early diagnosis and prognosis of major diseases such as tumors. Background technique [0002] microRNA (miRNA) is a kind of non-coding single-stranded small molecule RNA with a length of about 22 nucleotides, which widely exists in eukaryotic organisms such as animals, plants, and nematodes. miRNA degrades target mRNA or represses its post-transcriptional translation by specifically binding to the 3' untranslated region (3'UTR) of target mRNA, which ultimately leads to a decrease in protein synthesis of the target gene. miRNAs are involved in the regulation of all levels of life activities, including embryonic development, organ formation, cell proliferation, cell apoptosis, cell stress response, stem cell differentiation, endocrine regulation, immune regulation and the occurrence and development of diseases. A large numb...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q2600/178C12Q1/6853C12Q1/68C12Q1/6876C12N15/11C12Q2521/107C12Q2525/155C12Q2525/173C12Q2525/207C12Q2561/113
Inventor 苟德明康康
Owner 苟德明
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