Unit

UNIT 10.11 Preparation of RNA from Tissues and Cells

  1. Randall Ribaudo (hot phenol method)1,
  2. Michael Gilman (cytoplasmic RNA isolation and removal of contaminating DNA)2,
  3. Robert E. Kingston (CsCl isolation and poly(A) selection)3,
  4. Piotr Choczynski (single-step isolation)4,
  5. Nicoletta Sacchi (single-step isolation)5

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im1011s04

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Ribaudo, R., Gilman, M., Kingston, R. E., Choczynski, P. and Sacchi, N. 2001. Preparation of RNA from Tissues and Cells. Current Protocols in Immunology. 3:V:10.11:10.11.1–10.11.14.

Author Information

  1. 1

    National Institute of Allergy and Infectious Disease, Bethesda, Maryland

  2. 2

    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

  3. 3

    Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

  4. 4

    University of Cincinnati College of Medicine, Cincinnati, Ohio

  5. 5

    Life Technologies, Gaithersburg, Maryland

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: DEC 1992

Abstract

Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA.

The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A+) mRNAs from total RNA.