RNase III is a divalent metal dependent nuclease that cleaves long double-stranded RNA (dsRNA) into short dsRNAs (13-30 bases). Products of RNase III mimic siRNA structures produced by Dicer enzyme (5'-PO4, 3'-OH and a dinucleotide 3' overhang). This unique feature enable to generate a population of RNAs that, after transfection into mammalian cells, can induce RNAi (1-4).
Source: E.coli carring plasmid with rnc gene of E.coli RNase III.
Applications:
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Digestion of long dsRNA to short dsRNA.
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RNA structure studies (5).
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Transfection of RNase III cleavage products can be used to induce RNAi in mammalian cells.
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RNA processing and maturation studies (6-8).
Unit Definition: One unit of RNase III, E. coli, is the amount of enzyme required to digest 1 µg of dsRNA to siRNA in 20 minutes at 37°C in a total reaction volume of 50 µl.
Storage Conditions: Store at -20°C
Storage Buffer: 30 mM Tris-HCl, 500 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 50% glycerol, pH 8.0 at 25°C.
10 x Reaction Buffer: 300 mM Tris-HCl, 1.6 M NaCl, 10 mM dithiothreitol, 1 mM EDTA, pH 8.0 at 25°C.
Quality Control: All preparations are assayed for contaminating exonuclease, endonuclease, for nonspecific RNase and single- and double-stranded DNase activities. Typical preparations are greater than 90 % pure, as judged by SDS polyacrylamide gel electrophoresis.
References:
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Yang, D. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 9942-9947.
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Calegari, F. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 14236-14240.
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Donze, O. and Picard, D. (2002) Nucleic Acids Res, 30, e46. Laboratory, Cold Spring Harbour.
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Morlighem, J.E. et al. (2007) Biotechniques, 42, 599-606.
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Evguenieva-Hackenberg, E. and Klug, G. (2000) J. Bacteriol. 182, 4719.
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Nicholson, A. (1999) FEMS Microbiol. Rev. 23, 371.
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Drider, D. et al. (1999) J. Mol. Microbiol. Biotechnol. 1, 337.
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Grunberg-Manago, M. (1999) Annual Rev. Genet. 33, 193.
Reagent supplied: E1340-01: E1340-02:
10 x Reaction Buffer 750 µl 2 x 1.5 ml
MnCl2 0.5 M 200 µl 1.0 ml
RNase III 2 U/µl 100 µl 500 µl
RNase-free water 1.0 ml 3 x 1.5 ml
50 µl example reaction:
Assemble reaction on ice as follows:
5 µl 10 x Reaction Buffer
2 µl MnCl2 (final concentration in reaction 20 mM)
1-4 µg substrate RNA
1-2 µl RNase III
to 50 µl RNase-free water
Incubate 30-60 minutes at 37°C.
If cleavage products are used for transfection of mammalian cells, we recommend using EURx Universal RNA/miRNA Purification kit (Cat. No E3599) for purification of short RNA fragments in high quality.