Multiplex reverse transcription polymerase chain reaction combined with temperature gradient gel electrophoresis as a...

For the quantitation of intrinsic factor (IF) mRNA, an assay based on competitive reverse transcription and subsequent polymerase chain reaction (RT-PCR) combined with temperature gradient gel electrophoresis (TGGE) was established and validated with respect to precision and accuracy. IF-specific mRNA segments ("targets") were coamplified with known amounts of homologous "standard" RNA molecules, which differed from the targets by one base substitution. Following amplification, TGGE heteroduplex analysis proved to be a powerful method facilitating the efficient separation of these nearly identical target and standard DNA products. The measured absolute copy numbers of IF mRNA were put into relation to the constitutively expressed mRNA specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), quantified simultaneously by competitive multiplex RT-PCR. The resulting normalized IF mRNA expression rate in terms of n copies of IF mRNA/copy of GAPDH mRNA is independent of the mRNA heterogeneity and the abundance of specific transcripts within the RNA population of interest. Therefore, normalization relative to the housekeeping gene GAPDH provides a widely applicable value for comparative studies of gene expression on the level of mRNA. Here, a normalized IF mRNA expression rate of three copies per GAPDH mRNA molecule was measured in human stomach mucosa.