A method for purifying total mRNA from total RNA using slfn13

一种N端、样品的技术，应用在纯化总mRNA领域，能够解决mRNA质量和完整性难以保证、步骤繁琐、纯化效果不理想等问题

Active Publication Date: 2021-07-06
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects and deficiencies of the method for the total mRNA purified from total RNA in the above-mentioned prior art (the steps are cumbersome, the purification effect is not ideal and the quality and integrity of mRNA are difficult to guarantee, etc.), and a method for using SLFN13 method for purifying total mRNA from total RNA

Method used

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  • A method for purifying total mRNA from total RNA using slfn13
  • A method for purifying total mRNA from total RNA using slfn13
  • A method for purifying total mRNA from total RNA using slfn13

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Embodiment 1

[0034] Expression and purification of embodiment 1SLFN13

[0035] The N-terminal domain of SLFN13 (amino acid sequence 1-355 of human SLFN13, hSLFN13-N, Gene ID: 146857 and amino acid sequence 1-353 of rat SLFN13, rSLFN13-N, Gene ID: 303378; collectively referred to as SLFN13- N) Constructed into pET28 vectors respectively, among which "human SLFN13, Gene ID: 146857", "rat SLFN13, Gene ID: 303378" were derived from the GenBank database established by the National Center for Biotechnology Information (NCBI) in the United States. , transform the plasmid into the Rossetta (DE3) expression strain; pick a single clone and put it into 100ml LB medium with kanamycin and ampicillin double antibody for pre-cultivation. Transfer to 5L of TB medium supplemented with double antibodies and expand the culture at 37°C. When the OD reaches 0.4-0.6, cool down to 17°C and add 80uM IPTG to induce the expression of SLFN13 protein; after low-temperature induction for 16-20h, the bacteria are colle...

Embodiment 2

[0037] The present invention provides a method for purifying total mRNA from total RNA with SLFN13, the specific steps are as follows:

[0038] (1) Total RNA extraction: cells or tissue samples can use the traditional TRIzol-chloroform method to extract complete total RNA (if the sample is special, other applicable methods can be considered, and the quality and integrity of the total RNA should be ensured as much as possible);

[0039] (2) Digestion of tRNA and rRNA. According to the amount of total RNA extracted from the sample and the approximate content ratio of tRNA, rRNA and mRNA, calculate the amount of SLFN13 endonuclease and digestion time that need to be added; the content of tRNA, rRNA and mRNA in the total RNA is 12% respectively , 83% and 3% as examples, if take 10ul total RNA with a concentration of 1ug / ul, add 1ul 50uM SLFN13, add 2ul 10X enzyme digestion buffer, add 7ul ddH 2 O was made into a 20ul enzyme digestion system (that is, 5pmol SLFN13 was used for enz...

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Abstract

The invention discloses a method for purifying total mRNA from total RNA by using SLFN13, belonging to the field of biotechnology. The method for purifying total mRNA from total RNA with SLFN13 according to the present invention, the specific steps are as follows: (1) extraction of total RNA; (2) enzyme digestion of tRNA and rRNA in total RNA by SLFN13; (3) enzyme digestion After the end, the enzyme can be directly heated at 70°C for 15 minutes to inactivate the enzyme and obtain purified total mRNA. The present invention breaks the traditional concept of RNA purification, and introduces specific RNA endonuclease to enzymatically remove tRNA and rRNA that are planned to be removed from total RNA, which is simple and efficient. The method has the following advantages: low cost and easy acquisition; simple operation; time saving; good purification effect; and good for ensuring the stability of mRNA.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for purifying total mRNA from total RNA by using SLFN13 (Schlafen13). Background technique [0002] mRNA is a necessary stage of gene expression, which can reflect the transcription and expression information of a specific cell or tissue in a certain functional state, and is closely related to the characteristics and growth state of the cell. Transcriptome mRNA high-throughput sequencing is an efficient method that is currently favored. It only needs one test to quickly obtain the complete sequence information of RNA with poly-A tails, and can analyze gene expression, cSNP, new transcription, and new heterogeneity. Comprehensive transcriptome information such as variants, splice sites, allele-specific expression, and rare transcripts. The first important step in the sequencing experiment process is to extract the total RNA of the target cell or tissue and obtain high-qualit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806C12Q2521/301
Inventor 高嵩杨金玉谢伟邓翔宇
Owner SUN YAT SEN UNIV CANCER CENT
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