RT-PCR of SULT1E1 from rat hepatocytes
The RNA was isolated from hepatocyte of rats treated with DAS, Chalcone or their combination for 24, 48 and 72 hours. 1 μg of whole RNA utilized to perform reverse transcription PCR using Qiagen one step RT-PCR kit and SULT1E1 primer following the instructions provided in the kit. Total RNA concentration was measured by a NanoDrop Microvolume Spectrophotometers at 260 nm (A260 reading = 40 μg/ ml RNA) and its purity was tested from the ratio of A260/A280. The master mixprepared with stipulated proportions of ingredients including enzymes, dNTPs, template RNA and gene specific primer (both for human and rat SULT1E1) pairs (F 5’-CTTCCAGTATCATTTTGGGAAAAG-3’ and R 5’-TGGATTGTTCTTCATCTC-3’, all the primer specificity was satisfactory, that was tested by universal nucleotide alignment). To compare with control, 500 bp cDNA of rat β-actin and 200 bp cDNA of human β-actin were synthesized and loaded in the gel. The primer pair F 5′-GATGTACGTAGCCATCCA-3′/R 5′-GTGCCAACCAGACAGCA-3′ for the synthesis of rat β-actin and F 5′-GGCGGCAACACCATGTACCCT-3′/R 5′-AGGGGA GGGACTCGTCATACT-3′ for human β-actin were designed using the GeneFisher primer designing software and published earlier (20, 22). PCR was carried out in Eppendorf® Mastercycler® and the reaction program included reverse transcription at 50ºC for 30 min, PCR activation step 95ºC for 15 min, denaturation 94ºC for 1 min, annealing 64ºC for 1 min and extension at 72ºC for 1 min (30 cycles). Final extension was allowed for 10 min at 72ºC. PCR products were run an agarose gel electrophoresis image was captured by a BioRad gel doc system.