Validating Real-Time Quantitative RT-PCR Amplicons From Linearly Amplified Primitive Hematopoietic Progenitor mRNA

Objective. Low gene expression in rare cell subpopulations can make it difficult to identify transcripts using real time quantitative RT-PCR (qRT-PCR). Transcript number can be increased using linear amplification, but this technique amplifies the 3’ end of mRNA, imposing severe limitations on qRT-PCR primer design. Artifacts such as primer-dimers, introduced by these limitations, may interfere with accurate identification of genuine qRT-PCR amplicons. We explore the use of a sequential strategy to distinguish genuine amplicons from primer dimers. Methods. Cellular mRNA from CD34+/CD38-/lin- human umbilical cord blood (UCB) progenitors was linearly amplified using the RiboAmp Kit. qRT-PCR was performed using 3’ primers to assay bone morphogenetic protein (BMP) signaling cascade expression. PCR products were analyzed for their size, dissociation temperature and sequence specificity. Results. We found that 18/31 (58%) of primer pairs formed artifactual products due to primer dimers. Importantly, primer dimer prediction scores did not correlate well with the presence or absence of primer dimer formation under actual experimental conditions. Using a sequential strategy to generate a Primer Profile for each primer set, described here in detail for one gene (BMP-2) that was variably expressed under different culture conditions, we were able to distinguish genuine amplicons from primer dimers. Similar differences were seen between genuine amplicons and primer dimers for several other genes. Conclusions. These findings demonstrate the need for using such a strategy to detect false positive qRT-PCR results, particularly when using linear amplification of mRNA and SYBR Green.