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A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
Published on: December 2, 2009
A scintillation proximity assay for rna detection.
J Liu1, P A Feldman, J S Lippy
1DuPont Pharmaceuticals Company, Experimental Station, E400/5442, P.O. Box 80400, Wilmington, DE 19880, USA. jianwei.liu@dupontpharma.com
Analytical Biochemistry
|February 13, 2001
Summary
A new homogeneous scintillation proximity assay (SPA) detects RNA transcripts by using biotinylated oligodeoxynucleotides and scintillant beads. This method accurately measures RNA polymerase activity and aids in selecting antisense oligodeoxynucleotides.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Traditional methods for measuring RNA polymerase activity and probing RNA accessibility can be cumbersome.
- There is a need for homogeneous, high-throughput assays for RNA transcript detection.
Purpose of the Study:
- To describe a novel homogeneous scintillation proximity assay (SPA) for RNA transcript detection.
- To validate the assay's performance against traditional methods.
- To demonstrate its utility in antisense research.
Main Methods:
- Hybridization of 3H-labeled RNA transcripts with biotinylated oligodeoxynucleotides (ODNs).
- Binding of the hybrid to streptavidin-coated, scintillant-embedded beads.
- Detection of light emission from beads indicating bound RNA transcripts.
Main Results:
- The homogeneous SPA demonstrated strong correlation with traditional filter-binding methods for measuring RNA polymerase activity.
- The assay was successfully miniaturized to a 384-well format for high-throughput screening.
- The method effectively probed RNA-accessible sites for hybridization.
Conclusions:
- The developed homogeneous SPA is a sensitive and accurate method for detecting RNA transcripts.
- This assay is suitable for high-throughput screening and can be applied to antisense research for selecting effective antisense ODNs.
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