Accumulation of Cyclooxygenase-2 Gene Transcripts: MATERIALS AND METHODS(5)

4 Apr
2013

Ribonuclease protection assay (RPA). The RPAs were performed using a commercial kit (Ambion, Austin, TX) according to the instructions for the kit. Ten or twenty micrograms of total RNA of each sample was hybridized with the COX-2-specific or COX-1-specific cRNA probes and, as control for equal loading on the gel, bovine GAPDH-specific cRNA in the same vial. The differences in size of the protected fragments of COX-2 mRNA and GAPDH mRNA were sufficient to permit ready separation on a polyacrylamide gel. On the other hand, multiple bands in the protected fragments of both COX-2 mRNA and GAPDH mRNA would obscure the shorter COX-1 mRNA bands, which had to be done separately. The intensity of the protected fragments was quantified by densitometry (Advanced American Biotech Imaging, Fullerton, CA), and the results were expressed as a percentage of the intensity of COX-2 mRNA signal of the corresponding GAPDH mRNA. birth control yasmin

Statistical Analysis

The results of RPA assays, quantified by densitometry, were subjected to one-way ANOVA, followed by Newman-Keuls test. Differences in plasma PGFM concentrations between OT- and saline-treated cows were analyzed by twoway ANOVA for repeated measurements after logarithmic transformation of the data; they were also analyzed by linear regression and assessment of differences between slopes; p < 0.05 was considered significant.

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