Fig. 2

Obtaining comparable amounts of PCR products from transcripts of highly different abundances. a When samples of total RNA containing transcripts of genes A, B, and C that yield cDNAs of highly uneven concentrations, the amounts of some transcript-specific PCR products may be so low that differences between samples (e.g. induced vs. control) cannot be precisely quantified by capillary electrophoresis (as shown for transcript C). In order to allow reliable detection of the transcripts within the dynamic range of their differential regulation, the two samples (#1 and #2) are mixed (Step 1) and the concentrations of the gene-specific reverse primers (see Fig. 1) adjusted so that approximately equal amounts of cDNA fragments are obtained by RT-PCR from each of the transcripts (Step 2). Finally, the two samples, #1 and #2 are re-analysed with the adjusted primer concentrations (Step 3). Now, the x-fold change in the abundance of the transcripts of all genes can be determined due to the sufficient amount of their corresponding cDNAs (b)