title: Study on construction of cDNA libraries from rat normal liver and regeneration liver with smart technique.
abstract: The aim of the study is to construct cDNA libraries from the normal liver and regeneration liver of rat by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze their quality. The total RNA was separated from the normal liver and regeneration liver of rat and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo (dT) primer (contained sfi IB site) while the SMART oligonucleotide (contained sfi IA site) was utilized as a template so that the first-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR (long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction, enzyme. After cDNA size fractionation through Chroma Spin 400 column, the double-strand cDNA was ligated into the sfi I-digested lambda TripIEx2 vector and then the recombinant DNA was packagedin vitro. The unamplified rat normal liver cDNA library consists of 1.3×10(7) pfu/ml, and regeneration liver cDNA library consists of 1.6×10(7) pfu/ml in which the percentage of recombinant clones both are about 100%. Through testing, the high quality cDNA libraries containing full-length cDNA of rat normal liver and regeneration liver have been constructed. The titer of the amplified cDNA library is 4.5×10(10) pfu/ml and 3.6×10(10) pfu/ml. the average exogenous inserts of the recombinants both are about 1.5 kb. These results show that the normal liver and regeneration liver of rat cDNA libraries both have an excellent quality and lay solid foundation to study liver functions and the mechanism of liver regeneration.
authors.affiliation: Department of Cell Biology, Xinxiang Medical College, 453003 Xinxiang, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Jun-Tang
authors.forename: Wang
authors.initials: W
authors.lastname: Cong-Rui
authors.forename: Jogenananda
authors.initials: J
authors.lastname: Pramanik
authors.forename: Zhang
authors.initials: Z
authors.lastname: Hui-Yong
authors.forename: Feng
authors.initials: F
authors.lastname: Hui-Gen
authors.forename: Yang
authors.initials: Y
authors.lastname: Bao-Sheng
authors.forename: L I
authors.initials: LI
authors.lastname: Yu-Chang
authors.forename: Xu
authors.initials: X
authors.lastname: Cun-Shuan
keywords: LD-PCR; SMART; cDNA library; liver regeneration; rat