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

1 Apr


The six cows used for this experiment were paired according to gestation length; one cow in each pair was injected i.v. with 200 IU OT and the other with saline i.v. Two hours after OT injection the cows were exsanguinated, and a sample of intercaruncular endometrium was removed, frozen in liquid nitrogen, and stored at -75°C until processed for total RNA extraction and ribonuclease protection assay for COX-2 mRNA. Blood samples were collected at 15-min intervals for 30 or 45 min before and 90 min after the injection for measurement of plasma concentration of 13,14-hydroxy-15-keto-prostaglandin F2a (PGFM). The RIA for PGFM was performed with unextracted plasma as described and validated previously. The experimental protocol was approved by the Institutional Animal Care and Use Committee. buy cipro

RNA Extraction

In the course of these studies, total RNA was prepared by two methods: 1) tissues were pulverized in liquid N2 and homogenized in guanidium thiocyanate followed by ultracentrifugation through cesium chloride cushion as previously described or 2) tissues were processed according to Chomczynski and Sacchi using RNA-Clean kits (AGS, Heidelberg, Germany). All RNA samples were subjected to phenol/chloroform purification steps. Better yields were obtained with the former, more elaborate method, but the RNA quality, determined on electrophoresis on 1.2% agarose gels and optical density measurements at 260 nm and 280 nm, was equivalent with the two methods. Repeat experiments indicated virtually no within-sample variation, confirming the reproducibility of the data.