Transfer RNA reduces the formation of primer artifacts during quantitative PCR.

Recent advances have facilitated the real-time monitoring of polymerase chain reaction (PCR) by the inclusion of fluorescent labels, such as SYBR Green I (FMC BioProducts, Rockland, ME, USA), and have triggered the widespread use of quantitative PCR of cDNA targets (5–7). Nevertheless, because SYBR Green I binds nonspecifically to double-stranded (ds)DNA, it is essential to assess the specificity of product formation. Primer artifacts are commonly encountered when target concentrations are low, where mRNA quantities available can be precious or limited (3), difficult to extract (2) or might even constitute a rare component of the sample (4). Finding compounds that effectively reduce or eliminate the formation of primer artifacts is a major challenge in the field of fully quantitative PCR. Current methodology allows the identification of product differentiation through the analysis of DNA melting curves by plotting fluorescence as a function of temperature over the dissociation temperature of amplified products (5). The outcome of the analysis can result in several possible types of fluorescent signal, of which, only one is permissible for inclusion of quantitative PCR. Analysis is only permissible where exclusively the true product amplifies and thus results in the detection of a single sharp peak at the melting point specific to the amplified product (which is dependent on G/C content and product length). At lower concentrations, the signal derived from the artifact competes, in relative terms, Benchmarks