Description
ShortCut® RNase III, used with its manganese-containing reaction buffer, converts long double-stranded RNA into a heterogeneous mix of short (18–25 bp) interfering RNAs (siRNA) suitable for RNA interference in mammalian cells (1–3). 1.5 units (1 µl) of ShortCut RNase III is sufficient to convert 1 µg of dsRNA into siRNA suitable for RNA interference in mammalian cells.
ShortCut® RNase III, used with magnesium in the provided reaction buffer, digests long, immunogenic, double-stranded RNA present in IVT reactions. While optimization of enzyme and magnesium in the reaction is strongly encouraged, 0.1U of ShortCut RNase III with 0.25 mM MgCl2 removes dsRNA from 70 µg of IVT product while maintaining ssRNA integrity in most cases.*
*Not recommended for highly structured templates. Verify integrity of ssRNA and dsRNA removal after each treatment.
ShortCut RNase III digestion of dsRNA: (A) Varying amounts of ShortCut RNase III were incubated with 2 µg of a 500 bp dsRNA for 20 minutes at 37°C in a 50 µl reaction. Digests were analyzed by 20% TBE polyacrylamide electrophoresis. Marker lane contains a mixture of 21 bp siRNA Marker and 100 bp DNA Ladder (NEB #N3231). (B) dsRNA fragments (1 kb and 175 bp) were digested with ShortCut RNase III. Digests were analyzed by 20% TBE polyacrylamide gel electrophoresis.
GFP Silencing in COS-7 Cells: COS-7 cells co-transfected in a 24 well plate with a plasmid expressing GFP in the absence (control) or the presence of 30 ng (4 nM) of GFP siRNA prepared using the ShortCut RNAi Kit. Cells were photographed 48 hours post-transfection.
Product Source
An E. coli strain containing a genetic fusion of the E. coli RNase III gene (rnc) and the gene coding for maltose binding protein (MBP).
- This product is related to the following categories:
-
RNases,
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Epitranscriptome Analysis,
Properties & Usage
Materials Required but not Supplied
- Nuclease-free Water (NEB #B1500)
- dsRNA removal protocol only
Unit Definition
One unit is the amount of enzyme required to digest 1 µg of fluorescently labelled 40 bp dsRNA to siRNA in 20 minutes at 37°C in a total reaction volume of 20 µl.
Reaction Conditions
1X ShortCut Reaction Buffer
Incubate at 37°C
1X ShortCut Reaction Buffer
50 mM Tris-HCl
1 mM DTT
50 mM NaCl
(pH 7.5 @ 25°C)
Usage Concentration
1X
Storage Buffer
500 mM NaCl
10 mM Tris-HCl
0.5 mM EDTA
1 mM DTT
50% Glycerol
pH 8 @ 25°C
Heat Inactivation
No Unit Assay Conditions
One unit 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.