A scintillation proximity assay for rna detection.
A homogeneous scintillation proximity assay (SPA) for detection of RNA transcripts is described. 3H-labeled RNA transcripts are hybridized in solution to biotinylated oligodeoxynucleotides (ODNs), which are then bound by streptavidin-coated, scintillant-embedded beads. Only bound 3H-labeled RNA transcripts are brought in close enough proximity to stimulate light emission from the beads. The results from this novel homogeneous assay correlated well with those obtained using the traditional filter-binding methods to measure RNA polymerase activity. The assay has been miniaturized to a 384-well format compatible with automated high-throughput screening. This SPA method has also been successfully used to probe RNA-accessible sites to hybridization, and thus should provide a useful tool for selecting effective antisense ODNs in antisense research.
Liu
J
J
DuPont Pharmaceuticals Company, Experimental Station, E400/5442, P.O. Box 80400, Wilmington, DE 19880, USA. jianwei.liu@dupontpharma.com
Feldman
P A
PA
Lippy
J S
JS
Bobkova
E
E
Kurilla
M G
MG
Chung
T D
TD
D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D012588:Scintillation Counting; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic
D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012965:Sodium Chloride; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin