Analysis of RNA by Northern and dot blot hybridization:
Equal amounts of RNA samples (10 |ig) were electropho-resed on denaturing agarose-formaldehyde gels and transferred to hybridization membranes (Boehringer Mannheim) by using the capillary elution technique (Northern blotting) or by immobilizing unfractionated RNA by dot blotting (Convertible Filtration Manifold System, Gibco BRL, Life Technologies Inc) . RNA was cross-linked by baking the membranes at 80°C for 2 h under vacuum. Membranes were prehybridized and then hybridized overnight at 65°C with rat NOS-II probe, which was prepared by Taq PCR as described above. Detection was performed with the colorimetric detection reagents nitroblue tetrazolium and x-phos-phate with DIG detection system (Boehringer Mannheim) according to the manufacturer’s protocol and The DIG System User’s Guide for Filter Hybridization. buy flovent inhaler
RT-PCR: RT-PCR was carried out on 1 |ig of total RNA with an EZ rTth RNA PCR kit (Perkin-Elmer, Norwalk, Connecticut, USA) following recommended conditions and by using the following pairs of gene-specific primers: 5′-tgg ctt gcc ctt gga agt ttc tc (forward) and 5′-tgt ctc tgg gtc ctc tgg tca aa (reverse) for NOS-II ; 5′-ggg cca ggg tga tga gct ctg (forward) and starter 5′-gag gca ccc caa ctc tgt g (reverse) for NOS-III ; and 5′-agc ggg aaa tcg tgc gtg (forward) and 5′-cag ggt aca tgg tgg tgc tgc c (reverse) for beta-actin . PCR was carried out in an automatic DNA thermal cycler (Perkin-Elmer). PCR products were size fractionated by electrophoresis on ethidium bromide-stained 2% agarose gels, and the expected single bands corresponding to the 384 base pair fragment of NOS-II, the 324 base pair fragment of NOS-III and the 308 base pair fragment of beta-actin were observed. The molecular weight marker M1 (pUC19/MspI, DNA-Gdansk, Gdansk, Poland) was used (bands 501/489, 404, 331, 242, 190, 147 and 111/110 base pairs long).