Hybridization of oligo(dT) to RNA on nitrocellulose

Abstract

Quantitation of mRNA immobilized on nitrocellulose filters is an essential aspect of some studies in molecular biology. Hybridization of oligo(dT)18 to the poly(A) tails of mRNA can be used to measure filter-bound mRNA and thus provides a basis for comparing abundance of specific mRNAs. Hybridization rate of 32P-labeled oligo(dT)18 in 0.75 M NaCl, 75 mM sodium citrate, pH 7 (5 x SSC) to immobilized RNA was maximal at 25 degrees C. Filters were fully hybridized under these conditions within 1 hr when the oligo(dT)18 concentration was 10 pmol/ml or higher. Salt dependence of the dissociation temperature (Td) of oligo(dT)18:RNA duplex on filters was described by the equation Td = 42 - 20log10[molar Na+] (degrees C). With stringent washing of the duplex (four 5-min washes in 2 x SSC at room temperature), oligo(dT)18 gave no signal with plasmid DNA, rRNA, or tRNA. We have found that oligo(dT)18 can be used to normalize signal strengths rapidly and conveniently from total or oligo(dT)-selected eukaryotic RNA.