Identification of differentially expressed genes in rat asthma model by suppression subtractive hybridization technology.
- Author:
Bo ZHONG
1
;
Hui-lian WANG
;
Shahzad MUHAMMAD
;
Qi-lan NING
;
Yan HAN
;
Xu-dong YANG
;
Fu-jun ZHANG
;
She-min LU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Asthma; genetics; DNA, Complementary; genetics; Female; Gene Expression Profiling; Gene Expression Regulation; Gene Library; Male; Nucleic Acid Hybridization; methods; Polymerase Chain Reaction; Rats; Rats, Inbred Strains
- From: Journal of Zhejiang University. Medical sciences 2009;38(4):362-369
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo identify differentially expressed genes related to asthma by using a rat model.
METHODSTotal RNA extracted from the asthmatic rats was taken as the tester and the total RNA from the control rats as the driver. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGEM-T Easy vector to establish the subtractive library. The library was amplified through E.coli transformation and positive clones of the transformants were screened. The white clones in selective medium from cDNA library were isolated and digested by EcoR I restriction endonuclease. Thirty-six positive clones were chosen randomly and sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank (NCBI).
RESULTSThere were more than 300 white clones in the cDNA library. The clones were sequenced and similarity search (http://www.ncbi.hlm.nih.gov/BLAST) revealed 4 known genes, 2 ESTs without homologous genes and 3 potential new gene fragments.
CONCLUSIONThe forward-subtracted cDNA library for differentially expressed in the lung of asthmatic rats has been successfully constructed and the interesting candidate genes related to asthma have been identified.
