melting dna strands as a result of which the fluorescence yield is decreased suddenly the fluorescence behavior is converted by a special software into melting curves which are then given graphically as the first negative derivative as a function of temperature u2212 df dt vs t see fig1 detection of the transcription factor hepatic nuclear factor hnf 3 gamma from cdna cultured hepatocytes of the rat after the isolation and culturing of hepatocytes of the rat for 48 hours the cells where harvested and rna was isolated and purified by means of standardized methods rna 2 u03bcg and random primer roche diagnostics are heated for 10 minutes at 70 u00b0 c in order subsequently to initiate the reverse transcription 60 minutes at 42 u00b0 c dntps 1 nm roche diagnostics rnase inhibitor 40 u stratagene and amv reverse transcriptase 20 u promega are pipetted into random primers and rna buffer solution promega the reaction is stopped by heating to 95 u00b0 c for five minutes subsequently the claimed pcr cocktail is added to a lightcycler capillary and 400 nm of the specific oligonucleotide primer and the corresponding copydna are pipetted in 6 times 100 pg at the end of a 15 denaturing phase of 95 u00b0 c the pcr reaction is started after that the following cycle is repeated 46 times 95 u00b0 c for 3 seconds then 57 u00b0 c for 8 seconds and more will 72 u00b0 c for 12 seconds.