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Method for preparing exogenous circular RNA and method for quantitatively determining endogenous circular RNA

An exogenous circular and exogenous technology, applied in the field of molecular biology, can solve problems such as lack of circular RNA reference genes

Active Publication Date: 2015-02-04
JIANGHAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problem of the lack of reference genes for circular RNA in the prior art, the embodiment of the present invention provides a method for preparing exogenous circular RNA and a method for quantitatively detecting endogenous circular RNA

Method used

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  • Method for preparing exogenous circular RNA and method for quantitatively determining endogenous circular RNA
  • Method for preparing exogenous circular RNA and method for quantitatively determining endogenous circular RNA
  • Method for preparing exogenous circular RNA and method for quantitatively determining endogenous circular RNA

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Embodiment 1

[0044] The embodiment of the present invention provides a method for preparing exogenous circular RNA, such as figure 1 As shown, the method includes:

[0045] Step 101: selecting an exogenous linear RNA gene;

[0046] Step 102: Cloning the exogenous linear RNA gene into a prokaryotic expression vector to obtain the cloned exogenous linear RNA gene;

[0047] Step 103: Reverse transcribing the cloned exogenous linear RNA gene in vitro to synthesize exogenous linear RNA;

[0048] Step 104: removing the triphosphate structure at the 5' end of the exogenous linear RNA, and synthesizing a hydroxyl group at the 5' end of the exogenous linear RNA;

[0049] Step 105: performing phosphorylation modification on the exogenous linear RNA whose 5' end is hydroxyl, and synthesizing the exogenous linear RNA with phosphorylation modification at the 5' end;

[0050] Step 106: linking the 5' and 3' ends of the phosphorylated exogenous linear RNA at the 5' end to synthesize exogenous circular...

Embodiment 2

[0077] The embodiment of the present invention provides a method for quantitative detection of circular RNA, such as figure 2 As shown, the method includes:

[0078] Step 100: Using the method provided in Example 1 of the present invention to prepare exogenous circular RNA.

[0079] Step 200: performing quality control on the exogenous circular RNA.

[0080] Step 300: adding exogenous circular RNA to the total RNA of the sample to be tested and the control sample, the mass ratio of total RNA to exogenous circular RNA is 2000:1, to obtain a mixture of exogenous circular RNA and total RNA.

[0081] Step 400: Using real-time quantitative PCR to detect the contents of the target circular RNA and the exogenous circular RNA in the mixture of the exogenous circular RNA and the total RNA respectively.

[0082] Step 500: Using the exogenous circular RNA as a reference gene, determine the relative expression level of the target circular RNA in the sample to be tested.

[0083] Where...

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Abstract

The invention discloses a method for preparing an exogenous circular RNA and a method for quantitatively determining an endogenous circular RNA. The method for preparing an exogenous circular RNA comprises the following steps: cloning and multiplying an artificially synthesized DAN gene, transcribing the DAN gene into a linear RNA, modifying the tail end of the RNA, and connecting the head and the tail of the RNA to obtain the exogenous circular RNA. Exogenous circular RNA molecules are added to a total RNA according to a specific proportion to serve as a reference gene for real-time quantitative determination of the RNA, and therefore the real-time quantitative determination of the endogenous circular RNA is realized for the first time.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for preparing exogenous circular RNA and a method for quantitatively detecting endogenous circular RNA. Background technique [0002] Most of the RNA (Ribonucleic acid, ribonucleic acid) in the organism is linear, but a small part of the RNA is connected end to end to form a circle, which is called circular RNA. In recent years, circular RNAs have rapidly become a research hotspot in the RNA world, because circular RNAs have been found to be closely related to important biological functions and diseases. In addition, high-throughput sequencing technology has been applied to the detection of circular RNAs, which has greatly accelerated the discovery of circular RNAs and the analysis of their functions. [0003] For RNA, its expression level is a very important attribute. Whether RNA is expressed and the amount of expression determines the biological function of RNA. Fo...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 彭海李利利陈利红高利芬张继方治伟章伟雄卢龙李甜甜周俊飞
Owner JIANGHAN UNIVERSITY
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