The oligomers used for the PCRs were 5′-AACA-GCTTCCCGCTGAAAC-3′ and 5′ -TCTGCACATAACT-CCTTGCC-3′ and corresponded to positions 22-41 (exon 1) and 291-272 (exon 3) of the bovine MCP-1 cDNA, respectively. Samples were heated to 94°C for 5 min and amplified for 30 cycles (93°C for 30 sec; 60°C for 30 sec; 72°C for 30 sec) using a Biometra Personal Cycler (Bio-metra, Maidstone, Kent, UK). The final 72°C incubation was continued for a further 5 min. Reverse transcriptase blanks (no RNA) and PCR blanks (no cDNA products) were included in each analysis. Products were visualized by ethidium bromide staining after electrophoresis on 4% agarose gels (NuSieve GTG Agarose; FMC Bioproducts, Rockland, ME). In all experiments, reverse transcriptase and PCR blanks were negative. birth control yasmin
Identities of the PCR product were confirmed by restriction endonuclease digestion and by DNA sequencing of representative samples. Quantification of the PCR products was performed using an NIH-Image analyzer (Bethesda, MD). RT-PCR for ATPase was used as an interassay control. The ATPase primers were 5′-ACGAACACCACT CCTGGATGAGC-3′ and 5′-CACGGACGTCTCCAGGCTGTGTA-3′, corresponding to positions 21-43 and 191-213 of a bovine plasma membrane calcium-pumping ATPase cDNA. Under the conditions described here, the amount of amplified MCP-1 and ATPase cDNA produced was proportional to the number of thermocycles of the PCR reaction and the mass of RNA added to the reverse transcriptase reaction.