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High-throughout transcriptome sequencing method for micro sample

A high-throughput, transcriptome technology, applied in the fields of biotechnology and molecular biology, can solve the problems that trace samples and rare samples cannot be applied to transcriptome high-throughput sequencing technology, and trace samples are difficult to meet the requirements of transcriptome.

Inactive Publication Date: 2014-03-26
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to overcome the problem that existing micro-samples are difficult to meet the requirements of transcriptome high-throughput sequencing technology, and provide a transcriptome high-throughput sequencing method for micro-sample, aiming to solve the problem that micro-sample and rare samples cannot apply transcriptome Difficulties of high-throughput sequencing technology

Method used

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  • High-throughout transcriptome sequencing method for micro sample
  • High-throughout transcriptome sequencing method for micro sample
  • High-throughout transcriptome sequencing method for micro sample

Examples

Experimental program
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Embodiment

[0027] Example: Using yak oocytes as a representative of micro samples to perform the micro transcriptome high-throughput sequencing method of the present invention

[0028] Micro total RNA extraction from yak oocytes

[0029] Use the commercially available RNeasy Micro Kit (Qiagen, Germany) kit to extract the total RNA of micro-sample, and make slight adjustments according to the kit instructions. The specific steps are as follows:

[0030] (1) Select 20 mature yak MII stage oocytes and put them into a 1.5ml centrifuge tube, add 350μl lysate RLT (add β-mercaptoethanol before use, the final concentration is 1%), vortex or pipette Mix well.

[0031] (2) Transfer the cells added with the lysate to the filter column QIA shredder spin column (the filter column QIA shredder spin column is placed in the collection tube), centrifuge at 12,000rpm (~13,400×g) for 2min, and collect the filtrate.

[0032] (3) Add 1 volume (usually 350 μl) of 70% ethanol and mix with a pipette.

[0033...

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Abstract

The invention discloses a high-throughout transcriptome sequencing method for a micro sample. The high-throughout transcriptome sequencing method comprises the following steps: extracting total RNA (Ribonucleic Acid) of the micro sample; sampling a eucaryon sample; amplifying and enriching micro RNA; carrying out relevant enrichment, and carrying out reverse transcription and corresponding amplification enrichment on extracted micro total RNA by adopting an SMART technology; constructing a cDNA (Deoxyribonucleic Acid) I11umina-solexa sequencing library; and after carrying out fragmentation on a cDNA layer generated by enriching, carrying out high-throughout sequencing by using I11umina Hiseq TM2000. The lowest initial sample nucleic acid amount needed for the method disclosed by the invention can be as low as 10ng, so that great sample nucleic acid initial mass requirements for conventional high-throughout transcriptome sequencing are avoided, and therefore, the method can be effectively applied to high-throughout transcriptome sequencing of the micro sample, and particularly has high application prospect for a rear sample and a special sample which cannot obtain a great sample size.

Description

technical field [0001] The invention belongs to the fields of biotechnology and molecular biology, and in particular relates to a transcriptome high-throughput sequencing method for trace samples and its application. Background technique [0002] Transcriptome refers to the sum of all RNAs transcribed and expressed by a specific tissue or cell under certain environmental or physiological conditions, including both protein-coding mRNA and various non-coding RNAs. The link between group biological function. Transcriptome research, as an important omics research in the post-genome era, not only provides important means and methods for the study of gene expression and regulation, but also provides an important path for the discovery of functional genes, which is the basis and starting point for the study of gene function and structure . [0003] Large-scale transcriptomic detection technology has been developed for more than 20 years, from the initial differential hybridizatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2535/122C12Q2531/113
Inventor 兰道亮李键熊显荣陈亚冰胡敏苏小珊钟珍龚蔚华
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES