Quantification of cDNA generated by reverse transcription of total RNA provides a simple alternative tool for quantitative RT-PCR normalization.

Quantitative reverse transcription PCR (RT-PCR) is typically used to assay transcript abundance (often generalized as “gene expression”) by measuring a specific cDNA level. The method is very sensitive and is suitable for a broad range of cDNA concentrations. Its reliability depends on, among other factors, appropriate normalization (for a review, see References 1 and 2). The preferred method of quantitative RT-PCR normalization uses housekeeping genes with presumably invariant levels of expression as internal controls. Housekeeping gene-based normalization corrects the measured transcript levels for variable starting RNA amounts and for differences in RT efficiency. However, as there are no universally applicable genes with invariant expression, it is necessary to carefully evaluate the expression of candidate reference genes for every particular experimental system. Normalization with suboptimal housekeeping genes may result in different estimated values and lead to erroneous interpretations (3). To avoid a bias caused by the expression fluctuation of a single reference gene, Vandesompele et al. (4) proposed computation of the correction factor from several internal controls. However, this approach may increase the cost and laboriousness of experiments significantly. Another approach derives the correction factor for each sample from the input RNA amount, based either on spectrophotometric (A260), or on fluorometric estimation (5), meaning that there is no need to select a proper reference gene and verify its expression. However, this method relies upon the reproducibility of the RT reaction, which has been shown to be a major source of quantitative RT-PCR variation (6). Several other normalization strategies have been reviewed recently (7), none of them being used frequently. In this paper, we present a method of quantitative PCR normalization using the total amount of cDNA generated during RT. Our approach controls for the amount of starting material and variation of transcription efficiencies across RT reactions. Volkov et al. used PicoGreen® for quantifying RNA-DNA hybrid molecules after RT with poly(A)+ RNA added as a template (8). PicoGreen is a fluorescent dye that preferentially binds to double-stranded nucleic acids—the fluorescence of RNA-PicoGreen complexes reaching up to 10% of double-stranded DNA (dsDNA)Quantification of cDNA generated by reverse transcription of total RNA provides a simple alternative tool for quantitative RT-PCR normalization