Quantitation of Gene Expression by RT-PCR and HPLC Analysis of PCR Products

The quantitative measurement of specific mRNA species is of major importance for studies on gene expression. Northern blotting is a relatively insensitive method requiring microgram amounts of RNA. It is time consuming and semi-quantitative at best. Because of the limitations of Northern blotting, various strategies have been developed for quantitation of cDNA by polymerase chain reaction (PCR)-based methods (1 –4 ). Competitive PCR, in which a synthetic segment is co-amplified together with the target segment, is one of these approaches. Besides adding more steps to the experimental protocol, competitive PCR is limited because the competitor and target sequences are not necessarily amplified with the same efficiency, which impairs reliable quantitation (5 ). Recently, real-time PCR, in which the generated PCR-products are quantified fluorimetrically after each cycle, has become widely used (6 ). However, determination of fragment size for positive fragment identification is not—at least not directly—possible with this method.