Quantification of specific mRNA by flatbed scintillation counting of dual-labeled dot blots.
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.
Potter
C G
CG
University of Oxford, John Radcliffe Hospital, UK.
Ramshaw
A L
AL
Stickland
J E
JE
Parums
D V
DV
McGee
J O
JO
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
D000199:Actins; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D013462:Sulfur Radioisotopes