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Methods Mol Biol. 2014;1173:147-55. doi: 10.1007/978-1-4939-0931-5_13.

Isolation of small interfering RNAs using viral suppressors of RNA interference.

Author information

1
Drosophila Genetics and Epigenetics, Université Pierre et Marie Curie 9, Quai St Bernard, Building C - 5th floor - Room 517, Boîte Courrier 24, 75252, Paris cedex 05, France.

Abstract

The tombusvirus P19 VSR (viral suppressor of RNA interference) binds siRNAs with high affinity, whereas the Flockhouse Virus (FHV) B2 VSR binds both long double-stranded RNA (dsRNA) and small interfering RNAs (siRNAs). Both VSRs are small proteins and function in plant and animal cells. Fusing a Nuclear Localization Signal (NLS) to the N-terminus shifts the localization of the VSR from cytoplasmic to nuclear, allowing researchers to specifically probe the subcellular distribution of siRNAs, and to investigate the function of nuclear and cytoplasmic siRNAs. This chapter provides a detailed protocol for the immunoprecipitation of siRNAs bound to epitope-tagged VSR and subsequent analysis by 3'-end-labeling using cytidine-3',5'-bis phosphate ([5'-(32)P]pCp) and northern blotting.

PMID:
24920367
DOI:
10.1007/978-1-4939-0931-5_13
[Indexed for MEDLINE]

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