PMC:13922 / 18924-20004 JSONTXT

{"project":"Colil","denotations":[{"id":"T26","span":{"begin":763,"end":765},"obj":"9570382"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Oestrogen receptor mRNA measurement\nTotal RNA was extracted with TRIzol reagent (Boehringer-Mannheim, Mannheim, Germany), dissolved in RNase-free water and quantified by spectrophotometry at 260-280 nm. Aliquots of 30 μg RNA/15 μl were electrophoresed through a 1% agarose formaldehyde gel, capillary transferred to a Hybond-N membrane (Amersham), and treated according to the manufacturer's instructions. An EcoRI fragment (1300 pb) of pOR3 used as an ER-α mRNA probe was from the American Type Culture Collection (Rockville, MD, USA). Blots were hybridized sequentially with [32P]-labelled ER cDNA probe (109 cpm/mg cDNA, produced by random priming [Boehringer Mannheim]). Prehybridization (4 h) and hybridization (18 h) were performed as described previously [33]. The membranes were then washed with sodium citrate solutions of increasing stringency, the last wash being performed in 0.3 × sodium citrate solutions containing 0.1% SDS. Blots were visualized by exposure of the membranes for 1 day to Kodak XAR-5 film in an autoradiography cassette with an intensifying screen."}