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Published on: February 13, 2013
[Preliminary screening of a reverse-subtracted cDNA library for differentially expressed genes in rat liver of
Ding-zhi Fang1, Bing-wen Liu, Tao Shen
1Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Objective:
To identify differentially expressed cDNA sequences for rat liver of prothrombotic state (PTS) by screening a reverse-subtracted cDNA library.
Methods:
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:
Five positive clones were found by differential screening. For 3 of the 5 sequences, known genes of high homology (Score>225 bits) in the GenBank DNA database were found. For the other 2 sequences, only genes of low homology (Score=56) or intermediate homology (Score=151) were found.
Conclusion:
Five cDNA sequences were found PTS related. 3 of them may represent the genes that are either identical or nearly identical to previously reported genes The other 2 sequences are most probably new sequences not existing in GenBank DNA database, which have been registered in the GenBank DNA database as new sequences.