Identification of newly transcribed RNA
Michael E Greenberg1, Timothy P Bender
1Harvard Medical School, Boston, Massachusetts, USA.
Current Protocols in Molecular Biology
|February 12, 2008
Summary
Identify newly transcribed RNA using the nuclear runoff transcription assay. This method involves in vitro transcription of isolated nuclei with labeled UTP and hybridization to immobilized cDNAs for transcript detection.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Newly transcribed RNA plays a crucial role in gene expression regulation.
- Accurate methods for quantifying nascent RNA are essential for understanding cellular processes.
- The nuclear runoff transcription assay is a powerful technique for measuring active gene transcription.
Purpose of the Study:
- To describe methods for isolating nuclei suitable for in vitro transcription.
- To detail the nuclear runoff transcription assay for identifying newly transcribed RNA.
- To present protocols for preparing DNA templates for transcript hybridization.
Main Methods:
- Isolation of nuclei using detergent lysis, Dounce homogenization, or sucrose gradient centrifugation.
- In vitro transcription of isolated nuclei with 32P-labeled UTP to synthesize labeled RNA.
- Hybridization of labeled RNA to cDNAs immobilized on nitrocellulose filters for transcript detection.
Main Results:
- Successful isolation of nuclei free from membranes and cytoplasmic debris.
- Generation of labeled RNA transcripts from isolated nuclei.
- Detection of specific RNA transcripts through hybridization to prepared DNA filters.
Conclusions:
- The nuclear runoff transcription assay provides a reliable method for assessing gene transcription rates.
- The described protocols enable efficient isolation of nuclei and preparation of hybridization filters.
- This technique is valuable for studying gene expression dynamics in various biological contexts.
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