title: Quantification of specific mRNA by flatbed scintillation counting of dual-labeled dot blots.
abstract: 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.
authors.affiliation: University of Oxford, John Radcliffe Hospital, UK.
authors.forename: C G
authors.initials: CG
authors.lastname: Potter
authors.forename: A L
authors.initials: AL
authors.lastname: Ramshaw
authors.forename: J E
authors.initials: JE
authors.lastname: Stickland
authors.forename: D V
authors.initials: DV
authors.lastname: Parums
authors.forename: J O
authors.initials: JO
authors.lastname: McGee
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D005374:Filtration / Q000295:instrumentation; D051379:Mice; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D012588:Scintillation Counting; D013462:Sulfur Radioisotopes
chemical_list: D000199:Actins; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D013462:Sulfur Radioisotopes