RNA library preparation method, sequencing method and kit

A library and sequencing technology, applied in the biological field, can solve the problems of many steps in library construction, high reagent cost, long time consumption, etc., and achieve the effect of simple and fast operation, lightening the burden of data analysis, and improving the coverage.

Pending Publication Date: 2021-03-16
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process of building a library generally takes one day, which is time-consuming, with many steps in building a library and high cost of reagents
At the same time, when the RNA sequencing library constructed based on conventional library construction operations is sequenced, the sequencing results contain two cDNA data, and because the process of PCR synthesis of double-stranded DNA is introduced during the library construction process, the data repetition rate is high and the amplification rate is high. bias and errors

Method used

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  • RNA library preparation method, sequencing method and kit
  • RNA library preparation method, sequencing method and kit
  • RNA library preparation method, sequencing method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. High temperature interruption of RNA

[0060] Cut 100ng of RNA into 100-300bp RNA fragments, the steps are as follows:

[0061] 1) Thaw 5xFS buffer (Thermo Fisher product number: 18064014) and mix it upside down. Prepare the reaction system according to Table 1:

[0062] Table 1

[0063] Control A Experiment B RNA (50ng / μL) 2μL 2μL 5xFS buffer 4μL 4μL wxya 2 o

2μL 2μL Total 8μL 8μL

[0064] 2) Interrupt the reaction at high temperature in a PCR instrument: lid (PCR hot lid) 105°C, 94°C for 7 minutes, immediately place on ice to cool;

[0065] 3) Immediately after the cooling reaction, the next step of inversion reaction is carried out.

[0066] 2. First Strand cDNA Synthesis

[0067] 1) In the interrupted reaction tube, prepare the reaction system according to Table 2:

[0068] Table 2

[0069]

[0070]

[0071] 2) Reaction conditions: 10 minutes at 25°C, 15 minutes at 42°C, and RNA / cDNA hybrid fra...

Embodiment 2

[0088] 1. High temperature interruption of RNA

[0089] Break 100ng of RNA into 100-300bp fragments, and the breaking steps are as follows:

[0090] 1) After thawing the reagents, mix them upside down, and prepare the reaction system according to Table 4:

[0091] Table 4

[0092] T1 (control) T2 (experimental) RNA (50ng / μL) 2μL 2μL Frag / 1st strand buffer (Tiangen NG308) 5μL 5μL wxya 2 o

3μL 3μL Total 10μL 10μL

[0093] 2) Perform high-temperature interruption reaction in a PCR instrument: Tiangen: 94°C for 10 minutes, lid at 105°C, immediately place on ice to cool;

[0094] 3) Immediately after the cooling reaction, the next step of inversion reaction is carried out.

[0095] 2. First Strand cDNA Synthesis

[0096] 1) In the interrupted reaction tube, prepare the reaction system according to Table 5:

[0097] table 5

[0098]

[0099] 2) Reaction conditions: 10 minutes at 25°C, 15 minutes at 42°C, and RNA / cDNA...

Embodiment 3

[0109] 1. High temperature interruption of RNA

[0110] Cut 100ng of RNA into 100-300bp fragments, the steps are as follows:

[0111] 1) Thaw 5xFS buffer (Thermo Fisher product number: 18064014) and mix it upside down. Prepare the reaction system according to Table 6:

[0112] Table 6

[0113]

[0114]

[0115] 2) Perform high-temperature interruption reaction in a PCR instrument: 94°C for 7 minutes, lid 105°C, immediately place on ice to cool;

[0116] 3) Immediately after the cooling reaction, the next step of inversion reaction is carried out.

[0117] 2. First-strand cDNA synthesis and linker addition reaction

[0118] 1) In the interrupted reaction tube, prepare the reaction system according to Table 7:

[0119] Table 7

[0120]

[0121] 2) 10 minutes at 25°C, 15 minutes at 42°C, and 15 minutes at room temperature. After the reaction, the RNA / cDNA hybrid fragment plus linker product was obtained, and the product hybrid fragment Th-1 and library Th-2 were res...

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Abstract

The invention relates to the technical field of biology, in particular to an RNA library preparation method, a sequencing method and a kit. The RNA library preparation method comprises the following steps of performing reverse transcription by taking RNA as a template to generate first chain cDNA, thereby obtaining an RNA/cDNA heterozygote; and adding a first preset sequence to at least one tail end of the RNA/cDNA heterozygote to form an RNA/DNA heterozygote, wherein the RNA/DNA heterozygote is an RNA library, and the first preset sequence is located at at least one tail end of the cDNA chain. The RNA library prepared with the method is simple and rapid to operate, the constructed RNA library can be directly used for sequencing, and the method has no double-stranded cDNA synthesis process, namely no cDNA amplification and enrichment process, so that bias and errors caused by PCR amplification and enrichment can be avoided. Meanwhile, the invention discloses the RNA sequencing method which is used for directly measuring cDNA sequence information to obtain RNA sequence information. The invention also discloses the kit for RAN testing, and the kit can be used for constructing the RNAlibrary and sequencing the RNA library.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing an RNA library, a sequencing method and a kit. Background technique [0002] RNA sequencing research is the basis of gene function and structure research. It can study gene function and structure at an overall level. With the development of sequencing technology, RNA sequencing can be used to conduct more in-depth and complete research on transcriptomes. [0003] In order to achieve RNA sequencing, it is usually necessary to convert RNA into cDNA and then perform sequencing, that is, first construct a cDNA library and then perform sequencing. The library construction before RNA sequencing not only directly affects the RNA detection time, but also affects the sequencing results. At present, RNA library construction is generally divided into total RNA library construction, mRNA library construction, and small RNA library construction. The main steps of the curr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C12N15/10C40B50/06
CPCC12Q1/6806C12Q1/6869C12N15/1096C40B50/06C12Q2521/107C12Q2525/191C12N15/10C12Q1/68
Inventor 甘广丽张萌李改玲李妍张娟曾立董沈丽婉骆明杰
Owner GENEMIND BIOSCIENCES CO LTD
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