Quantification of specific mRNA by flatbed scintillation counting of dual-labeled dot blots
Potter, C.G.; Ramshaw, A.L.; Stickland, J.E.; Parums, D.V.; McGee, J.O.
Biotechniques 15(4): 738-743
1993
ISSN/ISBN: 0736-6205 PMID: 8251177 Document Number: 411965
A dual-labeling technique was developed for direct quantification of specific mRNA using a flatbed liquid scintillation counter. This method simultaneously measures cpm of 32P- and 35S-labeled probes bound to RNA dot blots and subtracts counts due to nonspecific background radioactivity bound to the filter. Probes for T-cell receptor and beta-actin (as the internal standard) were hybridized both separately and simultaneously to RNA isolated from five different sources. There was concordance between the radioactivity measured from single- and dual-hybridizations for each combination of 35S- and 32P-labeled probes. This methodology directly quantifies specific mRNA sequences bound to membranes and has potential for measuring gene dosage, without the need for re-probing or densitometric analysis.