Quantification of Lipid-Related mRNAs by Reverse Transcription-Competitive Polymerase Chain Reaction

Investigation of the in vivo regulation of gene expression in human subcutaneous white adipose tissue (WAT) relies on the ability to estimate the changes in specific mRNA levels in biopsies taken before and after a designed intervention (i.e., diet, exercise, hyperinsulinemic clamp, and so on). Such study is limited by the size of the samples that could be taken, and by the high lipid content of adipose tissue (AT), which renders the isolation of total RNA difficult. However, it is possible to obtain highly pure RNA preparations from low amounts of AT, using commercially available kits that are based on the selective binding of RNA molecules on silica-gel supports. Nevertheless, the low yield in total RNA with fat tissue requires a highly sensitive method to quantify specific mRNA molecules, in order to estimate the expression levels of the genes of interest. An adequate and powerful method is the quantification of mRNAs by reverse transcription followed by competitive polymerase chain reaction (RT-cPCR), which relies on the addition of a known amount of an exogenous DNA molecule (called competitor) to the amplification mixture, after the reverse transcription step (1 –3 ). In this method, however, the efficiency of the reverse transcription reaction is not controlled. We have therefore determined experimental conditions that allow 100% efficiency of cDNA synthesis during the reverse transcription step (4 ). This is possible when using a specific antisense primer (see Note 1 ) and a thermostable reverse transcriptase to perform the reaction at elevated temperature (see Note 2 ).