Gilman, M. (2001) Ribonuclease protection assay. In: Current Protocols in Molecular Biology. Wiley InterScience.
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.
| Item Type: | Book Section |
|---|---|
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification Investigative techniques and equipment > assays bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > ribonuclease |
| CSHL Authors: | |
| Communities: | CSHL labs |
| Depositing User: | Matt Covey |
| Date: | 2001 |
| Date Deposited: | 22 Jan 2014 20:55 |
| Last Modified: | 22 Jan 2014 20:55 |
| URI: | https://repository.cshl.edu/id/eprint/29211 |
Actions (login required)
 |
Administrator's edit/view item |