Nucleic Acids Research, Vol 26, Issue 11 2511-2518, Copyright © 1998 by Oxford University Press
AL Hayward, PJ Oefner, S Sabatini, DB Kainer, CA Hinojos and PA Doris
The present studies demonstrate a theoretical and practical framework for
the accurate quantitation of gene expression in RNA extracted from
microscopic tissue samples. The approaches are developed around competitive
RT-PCR techniques. Assay performance has been examined and validated at
both the RT and PCR steps. Our analysis of RT transcription efficiency for
a number of native and competitor combinations shows that this property can
differ, even for very similar templates. However, this difference is
consistent and, once identified and measured, can be removed as an obstacle
to accuracy. Using mathematical modeling, we have examined the simulated
co-amplification of native and competitor templates in PCR. Useful insights
have emerged from such modeling which indicate that differences in initial
amplification efficiency and the rate of decay of amplification efficiency
during the reaction can rapidly lead to inaccuracy, even while the slope
and linearity of log plots of the competitor input and reaction product
ratios are close to ideal. Finally, we show here that competitive RT-PCR
reactions do not have to remain in the log-linear phase of PCR in order to
accomplish accurate and precise quantification. Using appropriate
competitors sharing primer binding sites and high internal sequence
similarity, identical amplification efficiencies are preserved throughout
the reaction. Reaction products, including heteroduplexes formed between
native and competitor templates as reactions progress to plateau, can be
identified and quantified accurately using the new technique of denaturing
HPLC (dHPLC). This analytical technique allows the accuracy of competitive
RT-PCR to be preserved beyond the linear phase. The technique has high
sensitivity and precision and target abundances as low as 100 copies could
be reliably estimated.
ARTICLES
Modeling and analysis of competitive RT-PCR
Institute of Molecular Medicine, University of Texas Health Science Center, 2121 West Holcombe Boulevard, Houston, TX 77030, USA.
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