Nucleic Acids Research 13:2929-2987. 6 2 3 2 © IR L Press Limited, Oxford, England
- B. S. Sproat, D.M
- U niersity of A rona on Jne
Oligo(dT)-cellulose has been used to isolate poly(A) containing mRNA from 50 to 100-fold quantities of ribosomal RNA(l). In the isolation of poly(A) minus mRNA or of low abundance species of poly(A)-containing mRNA the use of defined oligodeoxyribonucleotide affinity columns is preferable. We have prepared a fully deprotected oligodeoxyribonucleotide (5'GGGCGCGCATGATAATCT-3') still anchored to the insoluble support used for synthesis, and used it as an affinity matrix for isolation of rubella viral RNA from total RNAs isolated from infected cells. Isolated RNA was used for cDNA synthesis, giving 25% cloning efficiency as screened by the oligonucleotide as probe. Total cellular RNA was allowed to hybridize to the immobilized oligonucleotide at 25°C for 30 min in binding buffer (0.5M NaCl, lOmM Tris-HCl, pH 7.5, lmM EDTA) with occasional mixing in an 1.5ml microfuge tube. The unbound RNA was removed by centrifugation and washing with binding buffer. Hybridized RNAs were eluted with H,0 by incubating at 50°C for 5 min, followed by centrifugation.