Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR.

Gene expression analysis plays an increasingly important role in many fields of biological research. The recently developed real-time PCR quantification method has many advantages over the conventional quantifications in terms of accuracy, sensitivity, dynamic range, high-throughput capacity, and absence of post-PCR manipulations (1, 2). Sequence-specific fluorescence-labeled probes (e.g., TaqMan) have been considered as a standard detection format in many diagnostic and research applications (3, 4), but are not very well suited for quantification of a large number of different sequences, because a new and relatively expensive probe is generally required for each amplicon under investigation. We have therefore optimized and validated a reverse transcriptase PCR (RT-PCR) assay for accurate expression profiling using the double stranded DNA-binding dye SYBR green I, which is a much more economical alternative to quantify any given transcript in a reaction. Using such a generic dye, different PCR amplicons and/or nonspecific amplification products could accurately be distinguished by the generation of so-called DNA melting curves and first-derivative melting peaks (5). During initial one-step RT-PCR reactions, we observed extensive accumulation of primer–dimers (PD)