RNA analysis by nuclease protection

Current Protocols in Neuroscience
Marianna Goldrick, Donald Kessler

Abstract

Nuclease protection assays (S1 nuclease protection and RNase protection) are extremely sensitive procedures for detection and quantitation of mRNA species in complex mixtures of total cellular RNA. These assays are well suited for mapping positions of external and internal junctions in RNA, such as transcription initiation and termination sites and intron/exon boundaries, and to discriminate between closely related targets by using probes designed to span the regions where the related genes differ the most. Also, because the size of the probes used in nuclease protection assays is a variable chosen by the investigator, probes may be designed to protect fragments of different sizes. This feature permits the simultaneous analysis of several different mRNAs in the same total RNA sample. In this unit, a method is included for RNase protection of target mRNA sequences, including hybridization of the probe to the target sequence, details of the actual protection assay, and detection of reaction products. An alternative method is provided for performing the RNase protection assay on a microvolume scale, which is useful when there are many samples to be analyzed. Support protocols describe synthesis and gel purification of labeled RNA ...Continue Reading

References

Jul 25, 1992·Nucleic Acids Research·P Chomczynski
Jan 1, 1987·Methods in Enzymology·J J Lee, N A Costlow
Sep 11, 1985·Nucleic Acids Research·E T Schenborn, R C Mierendorf
Aug 11, 1993·Nucleic Acids Research·P C BrownJ A Silverman
Nov 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·E A BurtonK E Davies
Jun 29, 2000·The Journal of Biological Chemistry·N A BenkuskyS K England
Oct 9, 2002·Mammalian Genome : Official Journal of the International Mammalian Genome Society·Pamela TranRima Rozen

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