ABSTRACT
Reverse transcription (RT) followed by a polymerase chain reaction (PCR) represents the most powerful technology to amplify and detect trace amounts of mRNA (Heid et al., 1996; Lockey, 1998). To quantify these low abundant expressed genes in any biological matrix the real-time quantitative RT-PCR (qRT-PCR) is the method of choice. Real-time qRT-PCR has advantages compared with conventionally performed ‘semi-quantitative end point’ RT-PCR, because of its high sensitivity, high specificity, good reproducibility, and wide dynamic quantification range (Higuchi et al., 1993; Gibson et al., 1996; Orland et al., 1998; Freeman et al., 1999; Schmittgen et al., 2000; Bustin, 2000). qRT-PCR is the most sensitive and most reliable method, in particular for low abundant transcripts in tissues with low RNA concentrations, partly degraded RNA, and from limited tissue sample (Freeman et al., 1999; Steuerwald et al., 1999; Mackay et al., 2002). While real-time RT-PCR has a tremendous potential for analytical and quantitative applications in transcriptome analysis, a comprehensive understanding of its underlying quantification principles is important. High reaction fidelity and reliable results of the performed mRNA quantification process is associated with standardized pre-analytical steps (tissue sampling and storage, RNA extraction and storage, RNA quantity and quality control), optimized RT and PCR performance (in terms of specificity, sensitivity, reproducibility, and robustness) and exact post-PCT data procession (data acquisition, evaluation, calculation and statistics) (Bustin, 2004; Pfaffl, 2004; Burkardt, 2000).
