RNase III (E.coli)

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:

• Digestion of long dsRNA to short dsRNA.
• RNA structure studies (5).
• Transfection of RNase III cleavage products can be used to induce RNAi in mammalian cells.
• 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:

1. Yang, D. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 9942-9947.
2. Calegari, F. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 14236-14240.
3. Donze, O. and Picard, D. (2002) Nucleic Acids Res, 30, e46. Laboratory, Cold Spring Harbour.
4. Morlighem, J.E. et al. (2007) Biotechniques, 42, 599-606.
5. Evguenieva-Hackenberg, E. and Klug, G. (2000) J. Bacteriol. 182, 4719.
6. Nicholson, A. (1999) FEMS Microbiol. Rev. 23, 371.
7. Drider, D. et al. (1999) J. Mol. Microbiol. Biotechnol. 1, 337.
8. 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.