Limitations imposed by heteroduplex formation on quantitative RT-PCR

RT-PCR, a rapidly emerging technique for the detection of RNA, is being used by many investigators to quantify small amounts of RNA. Accurate quantification of RNA content has been facilitated by the use of competitive amplicons as internal controls. We demonstrate that losses in sensitivity and accuracy are associated with an internal standard having sequence similarity to the primary amplicon. Analysis of PCR products under non-denaturing and denaturing conditions provided evidence that these losses were associated with heteroduplex formation. Subsequent analysis of factors associated with heteroduplex formation provides insights for future development of competitive assays. Assay considerations that can minimize limitations associated with competitive PCR protocols are discussed.