Reverse transcriptase-Polymerase Chain Reaction

Total RNA was extracted from a pooled sample of five mature male kidneys using Tri Reagent?(Sigma). The cDNA was synthesized from 1 ?/SPAN>g of total RNA using the First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Amplification reactions were assembled using oligonucleotides based upon conserved regions in teleost AR (GeneBank accession numbers: AB012095, AB012096, AB017158, AB023960, AF121257, AB025361 and AF326200). These oligonucleotides were: forward (5'-GGGAAACAGAAATACCTGTGTG-3') and reverse (5'-CTCTGCAATCATCTCTGGAAAG-3'). Amplification was conducted for 40 cycles at 94癈 for 30 s, at 40癈 for 1 min and at 72癈 for 1 min using a PTC-200 Thermal Cycler (MJ Research, Waltham, MA, USA). Amplified products were ligated into pGEM^{?/SUP>-T (Promega, Madison, WI, USA) and recombinant plasmids were isolated using the Wizard?Plus SV Miniprep System (Promega). Cycle sequencing was performed using the DYEnamic ET Terminator Cycle Sequencing Premix Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The reactions were resolved on an ABI Prism?377 DNA Sequencer (Perkin-Elmer, Milano, Italy) and the data obtained were analyzed using EditView (Version 1.01) (Perkin-Elmer).}