Quantitative PCR: validation of the use of a multispecific internal control

Although RT-PCR has many advantages over RNA blotting methods, it can be difficult to obtain quantitative information, due to the enzymatic nature of the PCR amplification, where small variations in amplification conditions result in drastic changes in product yield. In addition, due to the consumption of the reaction components and generation of inhibitors, the amount of products generated plateaus during later stages of the reaction. These problems can be overcome by the use of internal controls in the PCR. In competitive PCR, a DNA fragment containing the same primer template sequences as the target is used to compete for primers annealing and amplification. PCR products are then distinguished by size, restriction sites or Southern blot analysis. Competitive PCR methods require that the target gene and competitive fragment amplify with equal efficiency; this is the case when the internal standard is as similar as possible to the sequence of interest. We have examined whether measurements using a multispecific internal control can also be performed beyond the exponential phase of reaction, with the aim of rendering the technique independent of the number of cycles used. This possibility has already been suggested for methods employing an internal standard with the same sequence as the cellular template, apart from the presence of either a small intron, a small deletion or a mutated restriction site. To address this question, we have used for our multispecific internal control two templates both different in size and nucleotides composition from the cellular template. One pair of primers, specific for mouse IFN gamma cDNA and multispecific internal control, was used to amplify a 280 bp and a 250 bp fragment, respectively, and the second pair of primers, specific for mouse IL10, to amplify two fragments of 400 and 250 bp, respectively.