Optimized method for constructing RNA high-throughput sequencing library and application of optimized method

A sequencing library and high-throughput technology, applied in the field of molecular biology, can solve the problems of experimental reagents, consumables, waste of time and manpower, unbalanced reverse transcription, deviation of sequencing results, etc., to save labor costs and save purification reagents , the effect of improving the quality

Inactive Publication Date: 2017-11-24
哈尔滨博泰生物科技有限公司
View PDF1 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such a library construction method includes three tedious purification steps, causing a great waste of experimental reagents, consumables, time and manpower
At the same time, random p...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Optimized method for constructing RNA high-throughput sequencing library and application of optimized method
  • Optimized method for constructing RNA high-throughput sequencing library and application of optimized method
  • Optimized method for constructing RNA high-throughput sequencing library and application of optimized method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0026] The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to the following examples.

[0027] In the following examples, unless otherwise specified, all are conventional methods in the art.

[0028] 1 Extraction of total RNA and purification of mRNA from Arabidopsis leaves

[0029] 1) Total RNA was extracted from Arabidopsis leaves using the Trizol method. For specific steps, refer to the third edition of Molecular Cloning;

[0030] 2) Purification of mRNA from Arabidopsis leaves Use the mRNA Purification Kit from TOYOBO Company to purify mRNA. For specific steps, please refer to the product manual.

[0031] 2 Arabidopsis mRNA fragmentation

[0032] 1) Take 3-7 μL Arabidopsis leaf mRNA (total content 100-500 ng) into a new RNase free 0.2mL EP tube, add 2 μL 5X RNase III reaction buffer and 1 μL RNase III, add RNase free ddH 2 O to a total volume of 10 μL;

[0033] 2) Gently mix the mixture o...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention belongs to the field of molecular biological techniques and application, and particularly relates to an optimized method for constructing an RNA high-throughput sequencing library and application of the optimized method. The optimized method for constructing the RNA high-throughput sequencing library is characterized in that the method adopts RNase III to break to-be-sequenced RNA, adopts RNA ligase to connect linkers and adopts reverse transcriptase and a high-fidelity DNA polymerase system to conduct one-step reverse transcription and labeling PCR to construct the high-throughput sequencing library. The method can be applied to construction of the RNA high-throughput sequencing library. According to the optimized method for constructing the RNA high-throughput sequencing library, RNA is directly adopted to add the linkers onto single-strands, the step of random primer amplification is omitted, the influences of random primers can be eliminated, and more comprehensive RNA information is obtained; and an one-step method is adopted to conduct reverse transcription and labeling PCR, intermediate steps are reduced, and the preparation efficiency of the RNA library and the sequencing quality are enhanced effectively.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an optimized method for constructing an RNA high-throughput sequencing library and its application. Background technique [0002] Next-generation sequencing (NGS) represented by the Illumina sequencing platform has been widely used in the fields of DNA sequencing and RNA sequencing. RNA-seq can sequence the RNA sequences of mRNA, small RNA, non-coding RNA, RNA virus, etc. using high-throughput sequencing technology in one experiment. [0003] Usually, the construction of an RNA sequencing library generally includes the following steps: mechanically or enzymatically fragmenting the RNA fragments to a certain length, purifying the fragmented RNA, reverse transcription using random primers, synthesizing the second strand of cDNA, repairing the end and adding A Adapters, purification, index PCR amplification and index PCR product purification. Such a library construction ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68C40B50/06
CPCC12Q1/6869C40B50/06C12Q2535/122
Inventor 裴熙祥孙孝政
Owner 哈尔滨博泰生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products