Method for screening differential expression genes of mammal X, Y sperms

A technology of differentially expressed genes and non-human mammals, applied in the field of genetic engineering, can solve the problems of time-consuming and laborious, difficult to obtain low-abundance differentially expressed genes, etc., and achieve the effect of short test time, complete concentration and simple operation

Inactive Publication Date: 2012-12-26
TARIM UNIV
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Problems solved by technology

However, a large amount of poly(A) mRNA and multiple time-consuming steps are required, and it is difficult to obtain low-abundance differentially expressed genes

Method used

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Embodiment 1

[0030] Embodiment 1 A method for screening non-human mammalian XY sperm differentially expressed genes, specifically comprising the following steps:

[0031] 1) Sorting X and Y sperm by flow cytometry;

[0032] 2) Separation and purification of X and Y sperm mRNA;

[0033] 3) Obtain the mRNA differentially expressed between Y sperm and X sperm by subtractive hybridization between Y sperm and X sperm;

[0034] 4) Obtain the mRNA differentially expressed between X sperm and Y sperm by subtractive hybridization between X sperm and Y sperm;

[0035] 5) The obtained differentially expressed mRNA was amplified by Super SMART technology, and a Y-X subtracted hybrid cDNA plasmid library was constructed, cloned, sequenced, and analyzed by bioinformatics.

[0036] The method of obtaining differentially expressed mRNAs from Y sperm to X sperm by subtractive hybridization of Y sperm to X sperm comprises the following steps:

[0037] 1) Add magnetic bead suspension coupled with Oligo dT...

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Abstract

The invention discloses a method for screening differential expression genes of mammal X, Y sperms, comprising the following steps of: sorting X, Y sperms by a flow cytometer; separating and purifying mRNA of the X, Y sperms; using subtractive hybridization of the Y sperms on the X sperms to obtain the differential expression mRNA of the Y sperms relative to the X sperms; using subtractive hybridization of the X sperms on the Y sperms to obtain the differential expression mRNA of the X sperms relative to the Y sperms; amplifying the obtained differential expression mRNA by employing the Super SMART technology, and constructing a Y-X subtractive hybridization cDNA plasmid library, cloning, sequencing and performing a bioinformatics analysis. The mRNA obtained by using the method is complete and has high purity, high concentration and small size; step-by-step leaching of the chromatography column type allows a complete adsorption between cDNA and mRNA; and the control cDNA on the chromatography column can be repeatedly used in the subtractive cDNA cloning reaction. In the mean time, the method provided by the invention has advantages of simple and sensitive operation, short testing time, and no pollution.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for screening non-human mammal XY sperm differentially expressed genes. Background technique [0002] Analysis of differentially expressed genes is an important method to obtain gene function information. The methods for analyzing differential gene expression in populations mainly include differential display (DD), representational difference analysis (RDA), suppression subtractive hybridization (SSH), cDNA microarray, expression Sequence tag comparative sequencing analysis, serial analysis of gene expression (SAGE), etc. [0003] Differential screening is an early differential gene screening method, the main principle of which is to screen target clones from cDNA libraries by nucleic acid probe hybridization. The sensitivity is low, and it is only effective for a small number of genes that can be expressed in a large amount in one type of cell, but not expr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/10C12Q1/68
Inventor 高庆华孙献莹韩春梅
Owner TARIM UNIV
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