Method for constructing chain specific RNA library and application

A specific and library technology, applied in the field of constructing strand-specific RNA libraries, can solve the problems of increased detection cost, consumption of reagents, cumbersome process, etc., and achieve the effect of saving manpower and overcoming complexity and tediousness

Active Publication Date: 2019-01-18
广东安科华南生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The whole process is very cumbersome and wastes manpower and time; at the same time, a large number of reaction steps also consume a large amount of reagents, which increases the detection cost

Method used

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  • Method for constructing chain specific RNA library and application
  • Method for constructing chain specific RNA library and application
  • Method for constructing chain specific RNA library and application

Examples

Experimental program
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Effect test

Embodiment 1

[0020] In this example, human cell RNA is used to construct a strand-specific RNA library, and the illumina sequencing platform is taken as an example to further illustrate the present invention. Experimental methods such as figure 1 Shown:

[0021] 1. Using Ribo-Zero from Illumina TM The Magnetic Gold Kit removes ribosomes from sample RNA, and the specific steps refer to the instructions.

[0022] 2. Concentrate the ribosome-removed RNA to 5 μL in a PCR tube, add 5 μL of fragmentation buffer, process on a PCR instrument at 94°C for 3 minutes, and place it on ice immediately.

[0023] 3. Add the following components to the above fragmentation reaction solution:

[0024]

[0025]

[0026] Run the following program in the PCR instrument:

[0027] step

temperature

time

1

25℃

10min

2

42℃

30min

3

70℃

15min

4

4℃

Keep

[0028] 4. Add the following components to the above reverse transcription reactio...

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Abstract

The invention relates to a new method for constructing a chain specific RNA library and especially relates to a method for constructing the chain specific RNA library and an application. The method comprises the following steps: performing fragmentation treatment on RNA, thereby acquiring a fragmented RNA; performing reverse transcription on the fragmented RNA and then adopting RNase H for digesting a RNA/DNA heterozygote; connecting a digestion product with a joint, thereby acquiring a connected product; performing PCR amplification on the connected product, thereby acquiring an amplificationproduct, namely, the chain specific RNA library. According to the invention, RNase H is adopted for digesting the RNA/DNA heterozygote and then double-end joint connection is directly performed, so as to acquire the chain specific RNA library; due to an optimized reaction system, the process from sample RNA to joint connection can be finished by only adding reaction reagents in turn in a tube; compared with the conventional method for constructing the chain specific RNA library, the method provided by the invention is capable of overcoming complexity and tediousness of process and saving a large amount of manpower, time and reagent cost.

Description

technical field [0001] The invention relates to a new method for constructing a strand-specific RNA library, in particular to a method and application for constructing a strand-specific RNA library. Background technique [0002] With the development of next-generation sequencing technology, more and more scientific researchers are focusing on genome sequencing analysis, transcriptome sequencing analysis and analysis of the relationship between transcriptome and proteome. As an important means of scientific research, RNA sequencing is more and more widely used. In particular, the construction of strand-specific RNA libraries plays an important role in the in-depth research of RNA. [0003] Usually, the construction of a strand-specific RNA library requires the following tedious steps: fragmentation of sample RNA to an appropriate length, purification of fragmented RNA, reverse transcription using random primers, addition of dUTP for second-strand synthesis, purification of c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869C12Q1/6806
CPCC12Q1/6806C12Q1/6869C40B50/06C12Q2535/122C12Q2525/191C12Q2521/501
Inventor 卢文翔王莹涂慧珍白云飞顾万君朱毅华郑卫国
Owner 广东安科华南生物科技有限公司
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