Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels

Abstract

Poly A containing RNA isolated from embryonic chick calvaria was transferred from 6% formaldehyde 0.75% agarose gels to diazobenzyloxymethyl paper and the paper then hybridized to either nick translated pro alpha 1 collagen cDNA clones, pCg1 or pCg54, or to the nick translated pro alpha 2 collagen cDNA clone, pCg45. From the mobilities of the bands hybridizing most strongly to each, pro alpha 2 collagen mRNA was shown to be slightly larger than pro alpha 1 mRNA; they are 5100 and 4900 nucleotides long respectively. pCg54 also hybridized weakly to two bands of lower mobility, corresponding to RNAs 6.4 and 5.6 kb long. Neither pCg54 nor pCg45 hybridized to type II procollagen mRNA in poly A containing RNA isolated from embryonic chick sterna.

Oligo-2',5'-adenylate Synthetase Activity in K562 Cell Lines Persistently Infected with Measles or Mumps Virus

“…Total cytoplasmic RNA (60 gg) was electrophoresed on formaldehydeagarose gels, blotted and hybridized to the 32p-labelled probe as previously described (Fujii et al, 1988 ;Rave et al, 1979;Sawada & Fujinaga, 1980). For detection of human 2-5AS RNA, we used a 32p-labelled 1.4 kb fragment from pSP25R (Shiojiri et al, 1986).…”

Molecular Analysis of the Pyrimidine Deoxyribonucleoside Kinase Gene of Wild-type and Acyclovir-resistant Strains of Varicella-Zoster Virus

“…Total cellular RNA was isolated from VZV-infected and uninfected MRC-5 cells by the methods of Chirgwin et al (1979) and Glisen et al (1974) as modified by Ostrove et al (1985). The RNAs were separated by electrophoresis through 6% formaldehyde-l.5% agarose horizontal slab gels (Rave et al, 1979), transferred to nitrocellulose paper and hybridized to a nick-translated [32p]dATP-labelled pUC8 plasmid (sp. act.…”

Genetic and Epidemiological Studies of Dengue Type 2 Viruses by Hybridization Using Synthetic Deoxyoligonucleotides as Probes

“…Hybridization. Purified RNA or unpurified RNA extracted from cell supernatants was denatured in 10 × SSPE (1-8 M-NaCI, 0.15 r~-sodium phosphate pH 7.7, 0.01 M-EDTA) containing 18.5~ (v/v) formaldehyde, for 15 min at 65 °C (Rave et al, 1979;White & Bancroft, 1982), blotted onto nitrocellulose (NC), and baked for 1 h at 80 °C (Meinkoth & Wahl, 1984). The NC was prehybridized in 5 × SSPE, 0.5~ SDS and 100 ~g/ml homochromatography mix l (Jay et al, 1974) for 15 min at room temperature.…”