Adenylyl Cyclase Messenger Ribonucleic Acid: RESULTS(1)

RESULTS(1)

We found expression of eight of nine subtypes of ad-enylyl cyclase in freshly isolated rat myometrial cells. Isoforms II-IX were identified by appropriately sized PCR products after RT-PCR of rat total myometrial RNA (Figs. 1 and 2). These products were identified in at least three experiments using RNA from different rat uteri. In all cases, the controls lacking reverse transcriptase or lacking cDNA did not exhibit PCR products, confirming that PCR products were arising from newly synthesized cDNA and not residual genomic DNA or contaminating cDNA. We did not identify adenylyl cyclase type I mRNA in fresh rat myometrial cells despite obtaining an expected PCR product from rat brain RNA (Fig. 1). buy levaquin online

To confirm that the PCR products obtained represented amplified adenylyl cyclase cDNA, products were gel purified, excised, and sequenced. For isoforms II, III, V, VI, and VIII, PCR products corresponded to the published sequences for these rat isoforms. For adenylyl cyclases VII and IX, the sequenced PCR products corresponded to the published sequences for mouse types VII and IX.

Fig1Adenylyl Cyclase
FIG. 1. Representative RT-PCR results for DNase-treated total RNA from fresh rat uterine muscle or rat whole brain amplified with primers specific for adenylyl cyclase (AC) isoforms I-IV. Lane 1, фX174RF DNA/Hae III molecular weight standards; lanes 2 and 3, total RNA from whole rat brain; lanes 4-11, total RNA from rat uterine muscle. Reverse transcriptase (R.T.) was present (+) (lanes 2, 4, 6, 8, and 10) or absent (—) (lanes 3, 5, 7, 9, and 11) during cDNA synthesis. PCR products for adenylyl cyclase type I were detected in rat brain (lane 2) but not rat uteri (lane 4). Adenylyl cyclase types II-IV were detected in rat uteri (lanes 6, 8, and 10), including two splice variants of type IV (lane 10). No PCR products were obtained when reverse transcriptase was omitted from the cDNA synthesis reaction (lanes 3, 5, 7, 9, and 11), indicating a lack of genomic DNA contamination after DNase I treatment.

Fig2Adenylyl Cyclase
FIG. 2. Representative RT-PCR results for DNase-treated total RNA from fresh rat uterine muscle amplified with primers specific for adenylyl cyclase (AC) isoforms V-IX. Lane 1, фX174RF DNA/Hae III molecular weight standards; lanes 2-11, total RNA from rat uterine muscle. Adenylyl cyclase types V-IX were detected in rat uteri (lanes 2, 4, 6, 8, and 10). No PCR products were obtained when reverse transcriptase was omitted from the cDNA synthesis reaction (lanes 3, 5, 7, 9, and 11), indicating a lack of genomic DNA contamination after DNase I treatment.

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