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Indexes for digital gene expression profiling (DGE) and use method thereof

A gene expression profiling and tag technology, applied in the fields of tags and their using methods, second-generation sequencing technology, and solexa sequencing technology, can solve the problem of low repeatability of experimental results, unreliable data results, and inability to truly reflect sample-related information, etc. problems, to achieve the effect of increasing sequencing throughput and reducing costs

Active Publication Date: 2012-04-11
BGI TECH SOLUTIONS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Using the same RNA sample to construct a DGE library, if there is a problem of bias in the data output, it will lead to unreliable data results, cannot truly reflect the relevant information of the sample, and will also lead to low repeatability of the experimental results

Method used

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  • Indexes for digital gene expression profiling (DGE) and use method thereof
  • Indexes for digital gene expression profiling (DGE) and use method thereof
  • Indexes for digital gene expression profiling (DGE) and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1, the specific example of the construction of DGE label library

[0094] Using mouse liver RNA as a material, the 12 different DGE tag adapters 2 shown above were used for experiments, and a total of 12 mouse expression profile tag libraries with different tags were constructed

[0095] A preparation of mouse total RNA

[0096] 1. Take 4ug mouse liver total RNA in a 200ul PCR tube, dilute to 50ul with DEPC water, and mix well;

[0097] 2. Place the sample on a PCR instrument for denaturation at 65°C for 5 minutes to open the secondary structure;

[0098] 3. Place the sample on ice.

[0099] B Prepare GEX Sera-mag Magnetic Oligo(dT) Magnetic Beads

[0100] 1. Mix GEX Sera-mag Magnetic Oligo (dT) magnetic beads on a vortex mixer (vortex), and pipette 50ul into a 1.5ml non-stick EP tube;

[0101] 2. Place the EP tube on the magnetic stand for 2 minutes, and carefully suck out the supernatant;

[0102] 3. Add 100ul GEX Binding buffer to the EP tube, and mix...

Embodiment 2

[0247] Embodiment 2, the specific embodiment of the construction of DGE marker library

[0248] Using rice leaf RNA as the material, based on method 1, two tag libraries were constructed in parallel using Gex Index11 adapter2, and the data stability of the tag library output was tested.

[0249] A preparation of rice total RNA

[0250] 1. Take 4ug of rice leaf total RNA in a 200ul PCR tube, dilute to 50ul with DEPC water, and mix well;

[0251] 2. Place the sample on a PCR instrument for denaturation at 65°C for 5 minutes to open the secondary structure;

[0252] 3. Place the sample on ice.

[0253] B Prepare Sera-mag Magnetic Oligo(dT) magnetic beads

[0254] 1. Mix GEX Sera-mag Magnetic Oligo (dT) magnetic beads on a vortex mixer (vortex), and pipette 50ul into a 1.5ml non-stick EP tube;

[0255] 2. Place the EP tube on the magnetic stand for 2 minutes, and carefully suck out the supernatant;

[0256] 3. Add 100ul GEX Binding buffer to the EP tube, and mix the magnetic ...

Embodiment 3

[0377] Embodiment 3, the construction of DGE label library

[0378] Using Arabidopsis leaf RNA as material, four tag libraries were constructed based on method 1, and the data stability among the tag libraries was analyzed.

[0379] A Preparation of Arabidopsis total RNA

[0380] 1. Take 4ug of Arabidopsis total RNA in a 200ul PCR tube, dilute to 50ul with DEPC water, and mix well;

[0381] 2. Place the sample on a PCR instrument for denaturation at 65°C for 5 minutes to open the secondary structure;

[0382] 3. Place the sample on ice.

[0383] B Prepare GEX Sera-mag Magnetic Oligo(dT) Magnetic Beads

[0384] 1. Mix GEX Sera-mag Magnetic Oligo (dT) magnetic beads on a vortex mixer (vortex), and pipette 50ul into a 1.5ml non-stick EP tube;

[0385] 2. Place the EP tube on the magnetic stand for 2 minutes, and carefully suck out the supernatant;

[0386] 3. Add 100ul GEX Binding buffer to the EP tube, and mix the magnetic beads carefully;

[0387] 4. Place the EP tube on ...

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Abstract

In the invention, a unique index sequence (index1-12) is designed aiming at a digital gene expression profiling (DGE) library sample building method based on a Solexa Single End sequencing platform provided by the current illumina company, and indexes are embedded in a 3' adapter in a DGE library by adapters, thus successfully establishing a DGE index library building method. The method is suitable for building the DGE index library of any eukaryote RNA sample and is successfully used for solexa sequencing, thus not only increasing the sequencing throughput of the DGE samples but also lowering the cost of solexa aiming at DGE sequencing.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to the technical field of digital gene expression spectrum. In addition, the present invention also relates to a tag and a method for using it, and a method for constructing a digital gene expression spectrum library using the tag technology. The method of the present invention is particularly suitable for the second generation sequencing technology, especially the solexa sequencing technology. Background technique [0002] Digital Gene Expression Profiling (DGE) uses next-generation high-throughput sequencing technology and high-performance computing analysis technology to comprehensively, economically and quickly detect the gene expression of a specific tissue of a species in a specific state. Digital gene expression profiling has been widely used in basic scientific research, medical research and drug development and other fields. [0003] The specific tags of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B40/06C40B50/06C12Q1/68G16B35/00
CPCC40B50/06C12Q1/6869C12N15/1065C40B40/06C40B50/02G16B35/00G16C20/60C12Q2525/119
Inventor 章文蔚张艳艳田方于竞龚梅花
Owner BGI TECH SOLUTIONS
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