UNIT 4.10 Identification of Newly Transcribed RNA
Published Online: 1 APR 2007
Copyright © 2007 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Greenberg, M. E. and Bender, T. P. 2007. Identification of Newly Transcribed RNA. Current Protocols in Molecular Biology. 78:II:4.10:4.10.1–4.10.12.
- Published Online: 1 APR 2007
- Published Print: APR 2007
Newly transcribed RNA can be identified using the nuclear runoff transcription assay. In this assay, isolated nuclei, free of membranes and cytoplasmic debris, are used in an in vitro transcription reaction in the presence of 32P-labeled UTP. The labeled RNA can then be hybridized to cDNAs immobilized on nitrocellulose. This unit describes three methods for isolating suitable nuclei by detergent lysis, Dounce homogenization, and centrifugation on a sucrose gradient. Two Support Protocols describe the preparation of nitrocellulose filter strips containing double-stranded and single-stranded DNAs, which are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.
- nuclear runoff transcription assay;
- isolated nuclei