Ovary-Independent Estrogen Receptor Expression: MATERIALS AND METHODS(4)

15 Jan


Northern Blotting

Northern hybridization was performed to confirm specificity of the ovine 0ER8 probe for porcine RNA. The sequence of this partial cDNA for the ovine ER shares significant homology with ERa of other species, but not with the recently cloned ER. Total cellular RNA was extracted from uteri using TRIzol Reagent (Life Technologies, Gaithersburg, MD) according to manufacturer’s instructions. RNA (15 jjLg) was solubilized in sample buffer (single-strength MOPS [3-[/V-morpholino] propanesulfonic acid], 50% formamide, 2.2 M formaldehyde, 25% glycerol, and 0.025% bromophenol blue), heat denatured, and elec-trophoresed through a 1.25% agarose gel (1.1 M formaldehyde, single-strength MOPS). RNA was transferred to Nytran Plus (Schleicher and Schuell, Keene, NH) by downward capillary blotting and UV cross-linked. ventolin inhalers

Antisense cRNA probe was produced by in vitro transcription with T7 RNA polymerase from the EcoRI-linearized 0ER8 template, incorporating [a-32P]UTP (specific activity: 800 Ci/mmol; ICN Pharmaceuticals, Costa Mesa, CA). Membrane was prehybridized for 4 h; probe was denatured in hybridization buffer for 5 min at 65°C and then added to the hybridization tube. The membrane was hybridized overnight at 55°C, rinsed three times in 0.1-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) with 0.1% SDS for 20 min at 68°C, wrapped in plastic film, and exposed to a BI Imaging Screen (BioRad) for 12 h. Radioactivity was localized using a GS-525 Molecular Imager System (Bio-Rad).