The inherent quantitative capacity of the reverse transcription-polymerase chain reaction.
The quantitative capacity of the reverse transcription-polymerase chain reaction (RT-PCR) is generally underestimated. In this study, PCR and RT-PCR products were amplified from serially diluted DNA and RNA templates, respectively, using a 35-cycle PCR. In the approximate 30- to 100-fold range of template input above the lower limit of detection, herpes simplex virus ICP27 RT-PCR product yield was dependent on the logarithm of template mRNA input (r2 = 0.99). Likewise, regression analysis indicated that yields of interleukin-12 p40, herpes simplex virus DNA polymerase, and interferon-gamma PCR products were dependent on the logarithm of template DNA input over 40- (r2 = 0.98), 60- (r2 = 0.96), and 100-fold (r2 = 0.99) ranges, respectively. This quantitative relationship appears to derive from the competition for reactants between specific PCR products and nonspecific primer-dimers that occurs at limiting concentrations of template. Although primer-dimers are not generally considered a common feature of PCR, 30 of 32 primer pairs tested in this study produced primer-dimer amplification in the absence of template. Because the coefficient of variation in replicate PCRs was typically 10-20% in the linear range, the precision of PCR was sufficient to measure 4-fold differences in template concentration. Thus, with statistically adequate sample numbers, an appropriate standard curve, and the inherent quantitative capacity of the method, differences in the abundance of a mRNA species are measurable by 35-cycle RT-PCR.
Halford
W P
WP
Department of Microbiology, University of Pennsylvania, 221 Johnson Pavilion, Philadelphia, Pennsylvania, 19104-6076, USA. halford@mail.med.upenn.edu
Falco
V C
VC
Gebhardt
B M
BM
Carr
D J
DJ
D000818:Animals; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018259:Herpesvirus 1, Human; D017874:Immediate-Early Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014709:Vero Cells
D017931:DNA Primers; C057120:ICP27 protein, human herpesvirus 1; D017874:Immediate-Early Proteins; D012333:RNA, Messenger