For bovine COX-1, the antisense probe was prepared by in vitro transcription from the Sp6 RNA polymerase promoter out of the PCR fragment. The probe for COX-1 had a length of 301 bases, which included the multiple cloning site of 82 bases and protected an RNA sequence of 219 bases. COX-2 fragment was inserted into Bluescript (Invi-trogen, Carlsbad, CA) vector and was prepared by in vitro transcription with T3 polymerase after linearization of the plasmid with HindIII. The antisense cDNA probe for bovine COX-2 had a length of 376 bases, which included the multiple cloning site of 102 bases and protected an RNA sequence of 274 bases. The method of preparation was similar to that for the bovine cCOX-1 probe. buy flovent inhaler
As control probe for the RPAs, a fragment of the cDNA for bovine glycer-aldehyde phosphate dehydrogenase (GAPDH) was prepared as previously described. The probe had a length of 305 bases, including the multiple cloning site of 56 bases. It was inserted into the pGEM-T-Easy vector and was prepared by transcription from the Sp6 polymerase promoter after the plasmid was linearized with NcoI. It protected a cRNA sequence of 249 bases. All probes were prepared in the presence of [a-32P]dCTP as described in the instructions to the commercial RPA kit and were subsequently purified by polyacrylamide electrophoresis on 5% gels. The labeled compound, [a-32P]dCTP, was obtained from New England Nuclear (Bad Hamburg, Germany).