AN IMPROVED QUANTITATIVE RT-PCR FLUORESCENT METHOD FOR ANALYSIS OF GENE TRANSCRIPTS IN THE STS-65 SPACE-SHUTTLE EXPERIMENT

We describe a reverse transcription polymerase chain reaction (RT-PCR) technique using fluorescent dUTP to examine changes in mRNA level in samples. In this procedure, the amplified product is identified by the fluorescent detection system in an automated DNA sequencer, and if th e MW of the DNA/RNA or the fluorescent dye is different, several sampl es can be analyzed in a single lane. The basis for this technique is s imilar to that of radiolabeled methods, and we applied this technique for the comparison of the expression level of the rat c-myc gene in os teoblasts exposed to microgravity and unit gravity conditions. Using t he fluorescent- and radiolabeled methods, the level of rat c-myc mRNA were compared quantitatively and the results demonstrated that the c-m yc expression level was not altered by microgravity. Therefore, this f luorescent RT-PCR technique is useful for gene expression analysis par ticularly when the samples are limited, such as in space flight experi ments. The method also allows for rapid assessment of mRNA changes in many samples simultaneously.