[Construction of a cDNA subtractive library of rat brain after repeated +Gz exposure].
Objective. To construct a cDNA subtractive library of rat brain after repeated + Gz exposures with suppression subtractive hybridization (SSH). Method. Wistar [correction of Wister] rats were randomly divided into control group and repeated +Gz exposure group. Using an animal centrifuge, control rats were exposed to +1 Gz and exposure rats were exposed to +10 Gz for three times, each for 1 min with 30 min interval in between. Brains were taken 6 h after the last centrifuge run and Poly (A) + RNA were isolated. Moreover, single-strand cDNAs and double-strand cDNAs were synthesized in turn. After Rsa I enzyme restriction, +Gz exposure rat brain cDNAs were divided into two groups and ligated to the specific adaptor 1 and adaptor 2R, respectively. Then +Gz exposure rat brain cDNAs were hybridized with the control rat brain cDNA twice and underwent nested PCR twice. The PCR product was ligated with T/A plasmid vectors to set up the subtractive library. Result. The cDNA subtractive library of rat brain after repeated +Gz exposures with high subtractive efficiency was set up successfully. Conclusion. The highly efficient cDNA subtractive library may provide a solid foundation for screening and cloning differentially expressed genes in rat brain after repeated exposures to +Gz.
Cai
Qing
Q
Department of Molecular Biology, General Hospital of the Air Force, Beijing, China.
Liu
Hong-jin
HJ
Lin
Kai
K
Jiang
Jian-dong
JD
Zhu
Mei-cai
MC
D000818:Animals; D001921:Brain / Q000502:physiology*; D002498:Centrifugation; D018076:DNA, Complementary; D015723:Gene Library; D018471:Hypergravity; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar
D018076:DNA, Complementary