The components listed above were mixed and incubated at 42°C for 2 min before the addition of 200 units of Superscript I reverse transcriptase (Gibco BRL) and further incubated for 50 min at 42°C. Parallel reactions were performed as outlined above except that reverse transcriptase was not added to the reactions. These ‘‘ —RT controls’’ were then carried through to the PCR reaction to ensure that no PCR products were generated from sample tubes lacking reverse transcriptase, indicating a lack of genomic DNA contamination. The reaction was inactivated by heating to 70°C for 15 min. Complementary RNA was removed from the newly synthesized cDNA by the addition of 2 units of RNase H and incubation at 37°C for 20 min. Aliquots (1 ^l) of cDNA were either used immediately in 25-^l PCR reactions or stored at — 20°C for later use. buy asthma inhaler
PCR primers were used that would specifically amplify cDNA encoding adenylyl cyclase isoforms. For adenylyl cyclase isoforms II-VI, primers were used that have previously been shown to selectively amplify adenylyl cyclase isoforms from rat tissues. Adenylyl cyclase isoform I primers were designed on the basis of published bovine cDNA sequence, and rat brain total RNA was used as a positive control to ensure cross-species annealing of the PCR primers with rat adenylyl cyclase I. Adenylyl cyclase isoform VIII primers were designed on the basis of published rat cDNA sequence, and primers for isoforms VII and IX were designed based on published murine sequences. Type I primers were sense 5′-AGCACTTCCTA-ATGTCCAACCCT and antisense 5 ‘-CTAAGAAGTGCA-TCTCCTCCCAC, which correspond to bases 2651-2952 of the bovine type I adenylyl cyclase sequence.