Reverse Transcription and PCR

Reverse transcription and PCR were performed as follows.Total RNA from 100 μl of plasma was incubated in 20 μl containing 50 mmol/L Tris-HCl pH 8.3，150 mmol/L KCl.10 mmol/L MgCl2 10 mmol/L DTT，200 μmol/L dNTPs，0.1 μmol/L random hexamer，20 units“RNasin”（Promega Corp.，Madison，WI）and 5 units RT（Seikagaku America，Inc.，St.Petersburg，FL）at 37℃for 30 min.After heat heat inactivation of the reaction，the cDNA was amplified by PCR.Ten microliters of cDNA was amplified in 30 μl containing 50 mmol/L Tris-HCl pH 8.5，50 mmol/L KCl，2.5 mmol/L MgCl2，200 μg/ml gelatin，0.5 μmol/L primer pairs SK38/39 and 1 unit of Taq polymerase（perkin-Elmer Cetus，Norwalk，CT）.PCR was carried out for 30 cycles in a DNA thermal cycler（perkin-Elmer Cetus）with the following program：95℃for 30 s，55℃for 30 s，72℃for 60 s followed by a 10-min extension at 72℃at the end.All samples were run in duplicate.Negative controls（reagents alone）and positive controls（standard HNl RNA）were included for each set of amplification reactions.(https://www.daowen.com)