MRNA library construction method and kit

A kit and library technology, applied in the field of mRNA library construction, can solve the problems of time-consuming, complicated steps, and low library construction efficiency, and achieve the effect of improving library construction efficiency and efficiency

Active Publication Date: 2020-02-28
江西海普洛斯医学检验实验室有限公司
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Problems solved by technology

[0003] At present, the main process of constructing an mRNA-seq library includes enrichment of mRNA, and then fragmentation of mRNA, reverse transcription of one strand and two strands to synthesize double strands (cDNA at 16°C, 1h), and purification of cDNA products ( About 30min), followed by double-stranded cDNA end repair and A reaction (20°C, 15min; 65°C, 15min), adapter ligation, ligation product purification and fragmentation screening, PCR amplification and purification and other steps, however, this The construction process has problems such as complicated and time-consuming steps and low library construction efficiency

Method used

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  • MRNA library construction method and kit

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Embodiment 1

[0118] In this embodiment, the total RNA of the purified human leukocyte sample was extracted and purified using a commercial kit as an RNA sample, and then the RNA sample was quantified using a Qubit3.0 fluorescence quantitator, and the Agilent 4200 bioanalyzer was used to detect the completion of the sample, and finally a concentration of 216ng was selected. / μL and the RNA sample with a RIN of 10 was used as the RNA sample to be processed.

[0119] In this embodiment, the formulations of the first buffer solution, the first mixed enzyme solution, the second buffer solution and the third buffer solution are shown in Table 1 below. Wherein, dNTP is a mixture of equal amounts of dATP, dTTP, dCTP and dGTP. In addition, the first sequence of the Y-shaped linker is 5'-AATGATACGGCGACCACCGAGATCTACACATATGCGCACACTCTTTCCCTACACGACGCTCTTCCGATC-3', and the second sequence is 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTGATCGTATCTCGTATGCCGTCTTCTGCTTG-3'.

[0120] Table 1

[0121]

[0122] (...

Embodiment 2 to Embodiment 4

[0173] Compared with Example 1, Example 2 to Example 4 used the second mixed enzyme solution prepared according to the formula shown in Table 13, and the rest were processed in the same manner as in Example 1 until the mRNA library and library quality were obtained. The library quality inspection results are shown in Table 14.

[0174] Table 13 Second Mixed Enzyme Solution Formula

[0175]

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Abstract

The invention provides an mRNA library construction method. The mRNA library construction method includes mixing an mRNA fragment with a first mixed enzyme solution and a first buffer solution to obtain a first reaction system, and subjecting the first reaction system to first heat treatment to obtain a first reaction solution; mixing the first reaction solution with a second mixed enzyme solutionand a second buffer solution to obtain a second reaction system, and subjecting the second reaction system to second heat treatment to obtain a second reaction solution; mixing the second reaction solution with T4DNA ligase, a third buffer solution and a Y-shaped joint to obtain a third reaction system, subjecting the third reaction system to third heat treatment, and after the third heat treatment, performing purification to obtain a third reaction solution; adding a pre-mixed solution and a amplification primer into the third reaction solution to obtain a fourth reaction system, subjectingthe fourth reaction system to fourth heat treatment, and after the fourth heat treatment, performing purification to obtain an mRNA library. The mRNA library construction method and a kit, which are beneficial to improvement of the library construction efficiency, can be provided.

Description

technical field [0001] The present disclosure particularly relates to a method and a kit for constructing an mRNA library. Background technique [0002] Transcriptome sequencing (RNA-seq) is a sequencing technology that uses next-generation sequencing technology to comprehensively and rapidly obtain the sequence information and expression information of almost all transcripts in a specific cell or tissue in a certain state. Based on different research directions and strategies, transcriptome sequencing is divided into mRNA-seq for coding proteins and Ribo-zero RNA-seq for various non-coding RNAs. [0003] At present, the main process of constructing an mRNA-seq library includes enrichment of mRNA, and then fragmentation of mRNA, reverse transcription of one strand and two strands to synthesize double strands (cDNA at 16°C, 1h), and purification of cDNA products ( About 30min), followed by double-stranded cDNA end repair and A reaction (20°C, 15min; 65°C, 15min), adapter lig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12Q1/6806C12N15/1096C40B50/06C12Q2531/113C12Q2521/107C12Q2521/501C12Q2521/327C12Q2525/191
Inventor 刘江辉卢超马焕班吕艳花赖国荣
Owner 江西海普洛斯医学检验实验室有限公司
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