Adenylyl Cyclase Messenger Ribonucleic Acid: MATERIALS AND METHODS(9)

Type

VII primers were sense 5′-CCAGTTATTTAGAGAGA-GACCTG and antisense 5′ -CTTGCTCATCAGGGCCA-TGCTAA, which correspond to bases 3098-3657 of the murine type VII adenylyl cyclase sequence. Type VIII primers were sense 5′ -GGACAGCAGCTGGAGTACA-CAGC and antisense 5′ -CCTGATCCTTCAGGATGAGA-TAG, which correspond to bases 3513-4196 of the rat type VIII adenylyl cyclase sequence. Type IX primers were sense 5′ -AGCTTATCCTCACCTTCTTCTTCCTC and antisense 5′ -AGGACACGGTAGCACTCCTTGCC, which correspond to bases 3015-3327 of the murine type IX adenylyl cyclase sequence. buy ortho tri-cyclen online

PCR reactions (25 ^l) using 1 ^l of cDNA were assembled with the following (final concentrations) per reaction: 20 mM Tris, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.1 mM each dNTP, 1 ^M each primer, and 1.25 U Taq polymerase. PCR was carried out for 40 cycles as follows: 1) 94°C for 4 min, 2) 94°C for 1 min, 3) annealing temperature for 1 min, 4) 72°C for 2 min, 5) 39 cycles to step 2, 6) 72°C for 10 min. Annealing temperatures were 50°C for type I, 55°C for types II, VII, and VIII, and 65°C for types III, IV, V, VI, and IX. Two controls were included with each PCR experiment. The ‘‘—RT controls’’ were RNA sample tubes that lacked reverse transcriptase during the RT reaction to ensure that no PCR products were generated from contaminating genomic DNA despite DNase digestion. Secondly, PCR tubes were assembled that included all components except input cDNA to demonstrate the lack of nonspecific contamination during PCR. PCR products were analyzed by electrophoresis through 7% polyacrylamide gels with Фх DNA molecular weight markers and were visualized by ethidium bromide staining with UV transillumination. To confirm that PCR products represented the cDNA encoding specific adenylyl cyclase isoforms, representative PCR products were gel purified on 2-3% Nusieve (FMC Bioproducts, Rockland, ME) agarose gels, excised, electroeluted, and commercially sequenced using the same primers employed for PCR.

Statistics

Adenylyl cyclase activity increases by isoproterenol and inositol phosphate formation by oxytocin were analyzed by two-tailed, paired /-test. Values are expressed as mean ± SE; p values of less than 0.05 were considered significant.

This entry was posted in Myometrium and tagged Adenylyl Cyclase, Myometrium, Ribonucleic Acid.