Unit

UNIT 4.10 Identification of Newly Transcribed RNA

  1. Michael E. Greenberg1,
  2. Timothy P. Bender (isolation of nuclei)2

Published Online: 1 APR 2007

DOI: 10.1002/0471142727.mb0410s78

Current Protocols in Molecular Biology

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.

Author Information

  1. 1

    Harvard Medical School, Boston, Massachusetts

  2. 2

    University of Virginia, Charlottesville, Virginia

Publication History

  1. Published Online: 1 APR 2007
  2. Published Print: APR 2007

Abstract

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.

Keywords:

  • nuclear runoff transcription assay;
  • isolated nuclei