Unit

UNIT 4.7 Ribonuclease Protection Assay

  1. Michael Gilman

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb0407s24

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Gilman, M. 2001. Ribonuclease Protection Assay. Current Protocols in Molecular Biology. 24:II:4.7:4.7.1–4.7.8.

Author Information

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: OCT 1993

Abstract

Sequence-specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.