Use of competitive RT-PCR in the molecular analysis of peroxisome proliferation.
The technique of quantitative competitive RT-PCR to determine the levels of mRNA expression of genes encoding peroxisome proliferator-activated receptor alpha (PPAR alpha), acyl coenzyme-A (ACOX) and cytochrome P450 4A1 (CYP4A1) in primary rat hepatocyte cultures is described. This technique is based on the co-amplification of an internal standard (PCR MIMIC) and target DNA sequence with one set of primers. Following total RNA extraction and reverse transcription, competitive PCR was carried out by mixing various dilutions of known concentrations of PCR MIMIC with constant amounts of cDNA. Densitometry was then carried out on the DNA bands obtained following gel electrophoresis and, after correcting for size differences between the target DNA and MIMIC, the concentration of target DNA was calculated and expressed as attomoles (10-18 moles) per microgram total RNA. Constitutive levels of PPAR alpha, ACOX and CYP4A1 obtained were 0.037 +/- 0.003, 1.858 +/- 0.470m and 0.035 +/- 0.007 attomoles/microgram RNA, respectively. Following 24 h culture of rat primary hepatocytes in the presence of sodium clofibrate (a peroxisome proliferator), the levels of PPAR alpha, ACOX and CYP4A1 were increased by 2.1-, 3.3- and 12.8-fold, respectively. Thus the technique described in this study has high sensitivity and can be used to accurately measure the mRNA steady state levels in cell cultures.
Yaacob
N S
NS
Molecular Toxicology Group, School of Biological Sciences, University of Surrey, Guildford, UK.
Norazmi
M N
MN
Kass
G E
GE
Gibson
G G
GG
D042962:Acyl-CoA Oxidase; D000818:Animals; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D016373:DNA, Antisense / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D008830:Microbodies / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar
D017931:DNA Primers; D016373:DNA, Antisense; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D042926:Cytochrome P-450 CYP4A; D042962:Acyl-CoA Oxidase