CONSTRUCTION OF RNA STANDARDS FOR HIGH-RESOLUTION AUTOMATIC PRODUCT ANALYSIS IN QUANTITATIVE COMPETITIVE RT-PCR

The exponential character of PCR amplification may compromise quantita tive assays because it multiplies minor sample-to-sample variations. T o overcome these problems, several authors have used recombinant stand ard DNA or RNA molecules to be spiked into the samples in a dilution s eries of known copy numbers before co-amplification by PCR. To obtain an equal efficacy of reverse transcription and PCR amplification, stan dard and template molecules should be highly homologous. However the l imited resolution of commonly used agarose gel electrophoresis require s rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR. Due to a much higher resolu tion, automatic post-PCR analyzing systems based on laser-induced fluo rescence may help to overcome these difficulties. For using the capabi lities of these systems in quantitative competitive RT-PCR, we develop ed a protocol to construct uecornbinanr RNA standard molecules that on ly differ from the target sequence by a small deletion of 8 nucleotide s. It is based on PCR-induced mutagenesis and solid-phase in vitro tra nscription. This protocol was applied to quantify multidrug resistance gene (MDR1) mRNA in malignant cells, but it can easily,be adapted to any gene of interest.