Construction and preliminary screening of a forward-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state

• Published in: Sichuan da xue xue bao. Journal of Sichuan University. Yi xue ban Vol. 36; no. 6; p. 761
• Main Authors: Fang, Ding-Zhi, Liu, Bing-Wen, Shen, Tao, Bai, Huai
• Format: Journal Article
• Language: Chinese
• Published: China 01.11.2005
• Subjects: Animals; Base Sequence; DNA, Complementary - genetics; Gene Expression Profiling; Gene Library; Liver - metabolism; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Restriction Mapping - methods; RNA, Messenger - genetics; Sequence Analysis, DNA; Thrombophilia - genetics
• ISSN: 1672-173X

To construct and preliminarily screen the forward-subtracted cDNA library of differentially expressed genes in rat liver of prothrombotic state (PTS). 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 membr