[Construction and preliminary screening of a forward-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state]
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Published on: February 13, 2013
Ding-Zhi Fang1, Bing-Wen Liu, Tao Shen
1Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medical, Sichuan University, Chengdu 610041, China.
Objective:
To construct and preliminarily screen the forward-subtracted cDNA library of differentially expressed genes in rat liver of prothrombotic state (PTS).
Methods:
The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization using cDNAs synthesized from mRNA of PTS rat as Tester and cDNAs from mRNA of control rat as Driver. The products from the last PCR amplification of suppression subtractive hybridization were inserted into a T/A plasmid vectors to transform the Escherichia coli JM109 cells. To produce the library, the transformed cells were incubated at 37 C overnight on a LB agar plate containing ampicillin (50 microg/ml), IPTG and X-gal. Forward-subtracted cDNA probes and reverse-subtracted cDNA probes were prepared by nested PCR amplification, which were labeled with HRP. Positive clones were selected by differential screening in which forward-subtracted and reverse-subtracted cDNA probes were separately hybridized with the membranes slot-blotted by plasmid DNAs amplified and isolated from the library. Inserts in the positive clones were submitted to DNA sequencing. Nucleic acid sequence homology search was performed against the GenBank DNA database (non-redundant, and non-mouse and non-human EST entries) using the Standard nucleotide-nucleotide BLAST [blastn] program via a network connection to the National Center for Biotechnology information.
Results:
The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed. Two differentially expressed cDNA fragments were found after preliminary screening.
Conclusion:
The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed in the present study.