Quantitative multistandard RT-PCR assay using interspecies polymorphism

The use of RT-PCR to quantify mRNA is often compromised by the variability of reverse-transcription and amplification reactions as well as by the difficulty of assessing the amount and/or the integrity of input RNA. Use of a competitor RNA or the coamplification of an endogenous standard are widespread methods of monitoring these steps. Taking advantage of both sequence conservation between homologous genes in related animal species and interspecific polymorphism, a protocol that may be regarded as a compromise between these two methods is described here. Total RNA samples, extracted from even minute amounts of tissue belonging to a first animal species, were supplemented with a constant amount of total RNA prepared from a second animal species, which thus acts as a multistandard source. The mixture was reverse-transcribed using hexa-random primers. Separate PCRs were then undertaken so that, for each mRNA of interest, products from both origins could be distinguished. Since the ratio between amplified mRNAs is constant in the standard preparation, an accurate normalization in the assay samples of most variations inherent to PCR is obtained. This protocol allows quantification of several mRNAs species, whose amounts may be very different, in a single cDNA preparation.