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.