Analysis of rat cytochrome P450 isoenzyme expression using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).
A method was developed using reverse transcriptase-polymerase chain reaction (RT-PCR) to selectively detect and qualitatively determine the levels of mRNA expression of the major isoenzymes of cytochrome P450 (P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2, and 4A1) and fatty acyl-CoA oxidase (FACO) in the rat. Total liver RNA was isolated from male Sprague-Dawley rats treated with various inducers of cytochrome P450 (P450) and analyzed for the presence and relative quantities of each P450 isoenzyme mRNA using this technique. The specificity of the oligonucleotide primers used in the detection of each P450 mRNA was tested and confirmed through the simultaneous analysis of liver microsomal protein preparations for the presence of constitutive or inducible P450 apoprotein and enzyme activities using western immunoblotting and specific enzyme activity measures, respectively. This method of P450 expression analysis is proven to be highly specific and readily applicable for the assessment of P450 enzyme induction and down-regulation in the rat during routine toxicology studies when expression of the gene product is regulated by transcriptional activation and/or mRNA stabilization.
Morris
D L
DL
G. D. Searle & Company, Skokie, IL 60077, USA.
Davila
J C
JC
D000818:Animals; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers; D007527:Isoenzymes / Q000235:genetics*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System