RNA fragmentation reagent and application thereof

A reaction system and concentration technology, applied in the field of RNA sequencing, can solve the problems of slowing down the experiment speed, increasing the complexity of operation, and being unfavorable for automation

Active Publication Date: 2015-04-22
BGI TECH SOLUTIONS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This fragmentation buffer has a good effect on fragmenting RNA, but the more cumbersome steps affect the efficiency of the experiment. For example, a metal chelator must be added to terminate the fragmentation reaction. These steps not only slow down the experimental speed and increase the complexity of the operation, but also are not suitable for batch sample library construction, which is not conducive to the automation of the entire method process

Method used

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  • RNA fragmentation reagent and application thereof
  • RNA fragmentation reagent and application thereof
  • RNA fragmentation reagent and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0046] In order to determine the optimal RNA fragmentation conditions, the same RNA fragmentation reagent was used to test the same sample, 5 fragmentation temperature gradients were set as 70°C, 80°C, 90°C, 94°C and 100°C, and 6 fragmentation time gradients were 1.5min, 3min, 6min, 8min, 10min and 12min, a total of 30 programs.

[0047] 1.1 Sample reagents

[0048] MAQC-Agilent (Universal Human Reference RNA, UHRR, Stratagen); MAQC-AB (Human Brain Reference RNA, Ambion); 3X RNA fragmentation reagent: 200mM Tris-HCl (pH 8.0), 100mM KCl, 15mM MgCl 2 , 15mM ZnCl 2 (All components of fragmentation reagents were purchased from Sigma, and the reagents did not contain RNase); other reagent buffers, unless otherwise indicated, were from the mRNA-Seq sample preparation kit provided by Illumina.

[0049] 1.2 Experimental steps

[0050] 1.2.1 Purification of mRNA

[0051] 1) Take 1-10 μg of total RNA (MAQC-Agilent or MAQC-AB) into an RNase-free EP tube (Axygen), and dilute to 50 μl ...

Embodiment 2

[0090] 2.1 Sample reagents

[0091] 3X RNA fragmentation reagent: 100mM Tris-HCl (pH 8.0), 400mM KCl, 8mM MgCl 2 , 100mM ZnCl 2 (Each component of the fragmentation reagent is from Sigma, and the reagent does not contain RNase); the sources of other samples and reagent buffer are the same as in Example 1.

[0092] 2.2 Experimental steps

[0093] The test procedure is the same as 1.2.1~1.2.7.

Embodiment 3

[0095] 3.1 Sample reagents

[0096] 3X RNA Fragmentation Reagent: 350mM Tris-HCl (pH 8.0), 80mM NaCl, 100mMMgCl 2 , 8mM ZnCl 2 (Each component of the fragmentation reagent is from Sigma, and the reagent does not contain RNase); the sources of other samples and reagent buffer are the same as in Example 1.

[0097] 3.2 Experimental steps

[0098] The test procedure is the same as 1.2.1~1.2.7.

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Abstract

Provided are an RNA cleaving agent, a method for cleaving RNA, a method for constructing an RNA sequencing library, an RNA sequencing library and a sequencing method. The RNA cleaving agent comprises: a biological buffer suitable for maintaining the pH value of the RNA cleaving agent in the range of 7 to 9; and monovalent and divalent metal ions, the biological buffer and the monovalent and divalent metal ions are in effective concentrations.

Description

technical field [0001] The present invention relates to second-generation high-throughput sequencing, especially the field of RNA sequencing, and more specifically, the present invention relates to an RNA fragmentation reagent and its application. Background technique [0002] With the rapid development of next-generation high-throughput DNA sequencing technology, RNA sequencing (RNA-seq) has become an important new method for gene expression and transcriptome analysis. The transcriptome is what a specific cell can transcribe in a certain functional state. The sum of all RNAs, including mRNA and non-coding RNA. At present, the main process of RNA-Seq includes, firstly, using Poly(T) oligonucleotides to extract all RNAs with Poly(A) tails from total RNA, mainly the mRNA transcribed by coding genes, and the obtained RNA Randomly break into fragments, recover RNA fragments by ethanol precipitation, then use random primers and reverse transcriptase to synthesize cDNA fragments ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C40B50/06C40B40/08C12Q1/68
CPCC12Q1/6806
Inventor 章文蔚陈海燕胡轶霖田方张艳艳张秀清杨焕明
Owner BGI TECH SOLUTIONS
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