Retroscript first strand synthesis kit

Using our previously described methods,^{20 (link), 22 (link), 23 (link)} LV myocyte total RNA was extracted by RNAqueous^™ Phenol-Free Total RNA Isolation Kit (Invitrogen, Carlsbad, CA) and treated with RNase-free DNase I (Life Technologies, Grand Island, NY). The reverse transcription (RT) reaction was performed with RETROscript^™ First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA) using the antisense primers. The cDNA produced was amplified by polymerase chain reaction (PCR).
A 327 bp DNA fragment corresponding to rat β₁-AR coding region (bases 307 to 634) was produced by PCR with published the primers for rat β₁-AR: sense: 5′-GCCGATCTGGTCATGGGA-3′ (bases 307 to 324) and antisense: 5′-GTTGTAGCAGCGGCGCG-3′ (bases 617 to 634).^{24 (link)}A 308 bp DNA fragment corresponding to rat β₃-AR coding region (bases 12 to 319) was produced by PCR with the primers for rat β₃-AR: sense: 5′-ATG GCT CCG TGGCCTCAC-3′ (bases 12 to 29) and antisense: 5′-CCCAAGGGCCAGTGGCCAGTCAGCG-3′ (bases 295 to 319); and the 308 bp DNA fragment corresponding to rat β₃-AR was cloned into PCR 2.1 (Invitrogen, Carlsbad, CA). DNA sequencing of the recombinant plasmid was performed with T7 and M13 specific primers. The sequence of the fragment was 100% homologous to the corresponding region of the published rat β₃-AR cDNA (GenBank GI: 298306). GAPDH was co-amplified as an internal control.

Total RNA was extracted from cells or epidermal preparations using TRIzol reagent (Invitrogen) and 1μg was reverse transcribed using RETROscript first strand synthesis kit (Ambion). Real-time PCR was performed on a Bio-Rad CFX using iQ SYBR green Supermix (Bio-Rad). Primers used were: TFAM (5′-CCAAAAAGACCTCGTTCAGC-3′, 5′-ATGTCTCCGGATCGTTTCAC-3′), VHL (5′-TCCACAGCTACCGAGGTCAT-3′, 5′-CGACATTGAGGGATGGCACA -3′), HIF-1α (5′-GCGAGAACGAGAAGAAAAAGATGA-3′, 5′-ACTCTTTGCTTCGCCGAGAT-3′), HIF-2α (5′-GAACATGGCCCCCGATGAA-3′, 5′-AACCCAGTCTTGGCGTTCTC-3′), Epo (5′-AATGGAGGTGGAAGAACAGGCCAT-3′, 5′-CGAAGCAGTGAAGTGAGGCTACGTA-3′), BNIP3 (5′-GAAGCGCACAGCTACTCTCA-3′, 5′-TCCAATGTAGATCCCCAAGCC-3′), CAIX (5′-GCGCTAAGCAGCTCCATACT-3′, 5′-GCAGGGAAGGAAGCCTCAAT-3′), CTGF (5′-AGAACTGTGTACGGAGCGTG-3′, 5′-GTGCACCATCTTTGGCAGTG-3′), GLUT1 (5′-CGGCCTGACTACTGGCTTTG-3′, 5′-GCCAAACACCTGGGCAATAAG-3′), PAI1 (5′-GTAAACGAGAGCGGCACAGT-3′, 5′-GAGGATTGTCTCTGTCGGGT-3′), PGK1 (5′-TCTTGGGAGGCGCTAAAGTT-3′, 5′-AAGGCCATTCCACCACCAAT-3′), VEGF (5′-GGCCTCCGAAACCATGAACT-3′, 5′-CTGGGACCACTTGGCATGG-3′), mRPL19 (5′-GAAGGTCAAAGGGAATGTGTTCAA-3′, 5′-TTTCGTGCTTCCTTGGTCTTAGA-3′).