Construction method for short RNA fragment library, kit and application

A construction method and fragment technology, applied in the field of short RNA fragment library construction, can solve the problem that other nucleic acid sequences cannot be obtained

Active Publication Date: 2021-02-05
WUHAN BGI CLINICAL LAB CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These fragments may perform important functions outside the cell, but the current library sequencing process of short RNA fragments (small RNA) can only study the miRNA components, and other nucleic acid sequences cannot be obtained

Method used

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  • Construction method for short RNA fragment library, kit and application
  • Construction method for short RNA fragment library, kit and application
  • Construction method for short RNA fragment library, kit and application

Examples

Experimental program
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Embodiment 1

[0035] A small RNA library was constructed using human standard RNA sample UHRR (Agilent, Lot. #740000). The experiment is divided into 2 groups: the experimental group (using the construction method of the short RNA fragment library provided by the present invention, as attached figure 2 shown) and the control group (construction method of conventional short RNA fragment library); each group was set up with 3 replicates.

[0036] in, figure 1 Flowchart for routine small RNA library preparation. That is, in the process of constructing the Small RNA library in the control group, a 3' adapter is connected to the 3' end of the short RNA fragment; then a 5' adapter is connected to the 5' end of the short RNA fragment; in the reaction, since the ligase can only The RNA sequence at the 5' phosphate group and the 3' hydroxyl terminal is used as the reaction substrate, so only the miRNA components in the sample can be captured, and the capture efficiency of other short RNA fragment...

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Abstract

The invention relates to the field of gene sequencing, in particular to a construction method for a short RNA fragment library, a kit and application. The construction method comprises the following steps of carrying out pre-phosphorylation and dephosphorylation treatment on a short RNA fragment sample, so that a 3' terminal phosphate group in a short RNA fragment is modified into a hydroxyl group, and a 5' terminal hydroxyl group is modified into a phosphate group to obtain a first product; then connecting a 3' linker to obtain a first linker product; then carrying out dephosphorylation treatment to obtain a second product of which a 5' terminal is a phosphate group; then connecting a 5' linker to obtain a second linker product; and then carrying out reverse transcription and amplification to obtain the short RNA fragment library. The kit comprises T4 polynucleotide kinase and RNA 5' pyrophosphohydrolase. By utilizing the construction method and the kit, nucleic acid information of various short RNA fragments can be obtained; and the construction method and the kit are applied to the field of life science.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a construction method, kit and application of a short RNA fragment library. Background technique [0002] Extracellular RNA (Extracellular RNA or exRNAs) has extremely high research value in the field of life sciences, and its application in the field of liquid biopsy is a research hotspot. It is currently known that exRNAs include a variety of short RNA fragments, such as miRNA, siRNA, snoRNA, etc., and also include a large number of degraded fragments of long RNA (such as mRNA, long non-coding RNA, etc.). These fragments may perform important functions outside the cell, but the current library construction and sequencing process of short RNA fragments (small RNA) can only study the miRNA components, and other nucleic acid sequences cannot be obtained. [0003] Therefore, the library construction and sequencing process for short RNA fragment libraries needs to be further improved...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C40B50/06
CPCC12Q1/6806C40B50/06C12Q2521/525C12Q2525/191C12Q2525/117
Inventor 宗亮陈翠徐怀前田志坚
Owner WUHAN BGI CLINICAL LAB CO LTD
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