Unit

UNIT 4.10 Identification of Newly Transcribed RNA

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

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb0410s26

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Greenberg, M. E. and Bender, T. P. 2001. Identification of Newly Transcribed RNA. Current Protocols in Molecular Biology. 4:II:4.10.

Author Information

  1. 1

    Harvard Medical School, Boston, Massachusetts

  2. 2

    University of Virginia, Charlottesville, Virginia

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: APR 1994

This is not the most recent version of the article. View current version (1 APR 2007)

Abstract

Newly transcribed RNA can be identified using the nuclear runoff transcription assay. Isolated nuclei, free of membranes and cytoplasmic debris, are required for the assay. Cell lysis that does not allow the isolation of nuclei free of cell membranes and cytoplasmic material often results in poor incorporation of 32P-labeled UTP into nascent transcripts. Although there is no way to predict what cell types present this problem, many adherent cell lines and lymphocytes isolated from murine spleen or thymus do. Interestingly, very few nonadherent cell lines have posed this problem. As described in this unit, isolating nuclei by detergent lysis of cells works well for many tissue culture cell lines but may not be appropriate for all cell lines and many tissues. Protocols for detergent lysis and Dounce homogenization or cell lysis in an isoosmotic solution and centrifugation through a sucrose cushion are described along with a Support Protocol for the preparation of the cDNA nitrocellulose filter strips that are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.