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Method for detecting non-coding RNA transcription level based on hybridization connection method

A transcription level, non-coding technology, applied in the field of accurate measurement of non-coding RNA transcription levels, can solve the problems of unstable reverse transcription non-coding RNA efficiency and non-reproducibility, and achieve superior detection environment, reproducibility and accuracy The effect of increasing

Inactive Publication Date: 2015-05-06
HUNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Real-time fluorescence quantitative PCR is often combined with reverse transcription (RT-qPCR) and is widely used in the detection of non-coding RNA. However, due to the unstable efficiency of reverse transcription of non-coding RNA, many significant research results cannot be reproduced.

Method used

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  • Method for detecting non-coding RNA transcription level based on hybridization connection method
  • Method for detecting non-coding RNA transcription level based on hybridization connection method
  • Method for detecting non-coding RNA transcription level based on hybridization connection method

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Embodiment

[0055] a kind of like figure 1 The method for detecting non-coding RNA transcript levels based on the hybridization ligation method of the present invention as shown, comprises the following steps:

[0056] 1. Cell culture in vitro

[0057] HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai), and the cells were placed in DMEM medium (cell culture medium) containing 10% FBS (fetal bovine serum), 100 U / ml penicillin and 0.1 mg / ml streptomycin. At 37°C, 5% CO 2 cultured in a humidified incubator.

[0058] 2. Extraction of total RNA

[0059] The steps of RNA extraction by Trizol method are as follows: ①Take out the cells from the incubator, suck off the old culture medium, wash the cells quickly with 1×PBS preheated at 37°C, suck out the residual PBS, and then add 3~5mL of 37°C preheated 1 x PBS. ② Use a cell scraper to quickly scrape the cells, collect them in a 10mL centrifuge tube, put them in a refrigerated centrifuge (pre-cooling is...

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Abstract

The invention discloses a method for detecting non-coding RNA transcription level based on a hybridization connection method. The method comprises the following steps: extracting and purifying to obtain a sample solution containing to-be-detected target ncRNA; preparing a hybridization reaction system, so that the to-be-detected target ncRNA in the sample solution is hybridized with two complementary pairing DNA fragments to form an ncRNA-oligos hybrid chain; preparing a connection reaction system, and performing gap repair on the obtained ncRNA-oligos hybrid chain by using T4DNA ligase, thereby obtaining a to-be-detected sample containing HL-DNA; and taking the obtained to-be-detected sample containing HL-DNA as a template which is directly used for fluorescent quantitative PCR detection, and analyzing and detecting the transcription level of target ncRNA according to the detection result. The method disclosed by the invention is simple, convenient, rapid and safe, the to-be-detected non-coding RNA can be accurately and quantitatively analyzed, and the reproducibility of the experimental result is improved.

Description

technical field [0001] The invention relates to a method for accurately measuring the transcription level of non-coding RNA, in particular to a method for detecting non-coding RNA in combination with real-time fluorescent quantitative PCR. Background technique [0002] The traditional concept holds that the protagonist of RNA is the important components of gene expression such as mRNA, rRNA, and tRNA. The discovery of new functions of ncRNA has brought a new field of life science research. Now more and more studies have confirmed that RNA can be used in life activities. Play more different roles. In addition to gene transcription and protein synthesis, ncRNA is also involved in various RNA processes, DNA damage repair, and genome rearrangement. As more and more new ncRNAs are discovered and identified, they form an increasingly large biological network, which not only revolutionizes the traditional concept in the field of life sciences, but also shows infinite possibilities...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 周征曾珍王丹王子天陈润生
Owner HUNAN UNIV
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