How to correct for errors in mRNA quantitation by competitive PCR due to heteroduplex formation of amplification products.
The Q-RT-PCR method for determining mRNA levels is an internally-controlled quantitative reverse transcriptase linked polymerase chain reaction to assess gene activity by monitoring the accumulated stable transcript level, in a given sample of extracted total RNA. Internal and competitive standards are used for mRNA titration because of the exponential nature of PCR; possible variations in RT efficiency are corrected for by the use of riboprobe RNA standards. A renin mRNA assay has been optimized by altering PCR primer parameters. Q-RT-PCR suffers from the occurrence of heteroduplex DNA formation between amplification products from homologous standard and target mRNA. Co-migration of various PCR products is shown to occur on neutral agarose gels, and this will lead to serious errors in mRNA determinations. Adjustment of the standard electrophoresis conditions is required for absolute mRNA quantitation by Q-RT-PCR.
Boer
P H
PH
Cardiac Molecular Biology, University of Ottawa Heart Institute, Ontario, Canada.
Ramamoorthy
J
J
D000818:Animals; D004587:Electrophoresis, Agar Gel; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015347:RNA Probes / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis* / Q000592:standards; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D012083:Renin / Q000235:genetics; D015203:Reproducibility of Results
D009692:Nucleic Acid Heteroduplexes; D015347:RNA Probes; D012333:RNA, Messenger; D012083:Renin