**2.6 Reverse transcription-polymerase chain reaction (RT-PCR)**

Total RNAs of MH7A cells before and after treatment with resveratrol (100 M) were purified by an acid/guanidine/thiocyanate/chloroform extraction method using a Sepasol-RNA I Super kit (Nacalai Tesque, Kyoto, Japan). After purification, total RNAs were treated with RNase free-DNase I (2 unit) at 37°C for 30 min to remove genomic DNAs, and 10 g of RNAs were resuspended in water. Then, oligo dT primers, dNTP, 5 x First Strand buffer, and SuperScript III RNase H-Reverse Transcriptase were added to the RNA solution and incubated at 65°C for 5 min followed by 60°C for 1 min, 56°C for 60 min, 58°C for 60 min, 85°C for 5 min to synthesize the first strand cDNA. Subsequently, 1 l of the reaction solution was diluted with water and mixed with 10 x PCR reaction buffer, dNTPs, MgCl2, oligonucleotide, dimethylsulfoxide [final concentration, 5% (v/v)] and 1 unit of Taq polymerase (Fermentas, St. Leon-Roth, Germany) (final volume, 20 l). RT-PCR was carried out with a Takara Thermal cycler Dice (Takara Bio Inc.) programmed as follows: the first one step, 94°C for 2 min and the ensuing 30 cycles, 94°C for 1 s, 62°C for 15 s, and 72°C for 30 s using primers shown in Table 1. PCR products were stained with ethidium bromide and visualized by 2% agarose electrophoresis.


Table 1. Primers used for RT-PCR
