Home RNA Synthesis and Modification ShortCut® RNase III

ShortCut® RNase III
Gray property icon for NEBU recombinant Gray property icon for incubation temperature 37° Gray property icon for heat inactivation no

ShortCut RNase III converts long double-stranded RNA into a heterogeneous mix of short (18–25 bp) interfering RNAs (siRNA).

  • Isolated from a recombinant source
  • Useful for gene silencing and target validation
  • Supplied with 10X Reaction Buffer
  • Not sure which RNase to choose? Learn more about the different RNases offered by NEB.
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Catalog # Cat # Concentration Conc Size List Price List Price Your Price Quantity
M0245S 2,000 units/ml 200 units $157.00 $157.00
M0245L 2,000 units/ml 1,000 units $629.00 $629.00
Please enter a quantity for at least one size

  • ShortCut RNase III used with its manganese-containing reaction buffer,  converts long double-stranded RNA (dsRNA) into a heterogeneous mix of short (18–25 bp) interfering RNAs (siRNA) suitable for RNA interference (RNAi) in mammalian cells (6–8). It is useful for gene silencing and target validation and eliminates a trial-and-error approach with synthetic siRNA.

    ShortCut RNase III used with magnesium-containing buffer can also be used to significantly reduce dsRNA content in IVT reactions. Guidelines for optimizing this application can be found in our application note: Use of ShortCut RNase III for dsRNA removal from IVT samples

    Figure 1: ShortCut RNase III digestion of dsRNA

    Gels depicting ShortCut RNase III digestion of dsRNA at various amounts
    (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.


    Figure 2: GFP silencing in COS-7 cells

    GFP silencing with ShortCut® RNAse III
    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,
    Epitranscriptome Analysis

    • Reagents Supplied

      The following reagents are supplied with this product:

      NEB # Component Name Component # Stored at (°C) Amount Concentration
      • NEB # Stored at (°C)
        M0245S -20
        Component Name Component # Stored at (°C) Amount Concentration
        ShortCut® RNase III M0245SVIAL -20 1 x 0.1 ml 2,000 units/ml
        ShortCut Reaction Buffer B0245SVIAL -20 1 x 1 ml 10 X
        10X EDTA B0255AVIAL -20 1 x 1 ml 500 mM
        MnCl2 B0786AVIAL -20 1 x 1 ml 200 mM
        Glycogen RNase-Free B1564SVIAL -20 1 x 0.05 ml 10 mg/ml
      • NEB # Stored at (°C)
        M0245L -20
        Component Name Component # Stored at (°C) Amount Concentration
        ShortCut® RNase III M0245LVIAL -20 1 x 0.5 ml 2,000 units/ml
        ShortCut Reaction Buffer B0245SVIAL -20 1 x 1 ml 10 X
        10X EDTA B0255AVIAL -20 1 x 1 ml 500 mM
        MnCl2 B0786AVIAL -20 1 x 1 ml 200 mM
        Glycogen RNase-Free B1564SVIAL -20 1 x 0.05 ml 10 mg/ml

    • Properties & Usage

      Materials Required but not Supplied

      • Nuclease-free Water (NEB #B1500)
      • EDTA
      • Optional (for ethanol precipitation)
        • 3 M NaOAc (pH 5.5)
        • RNase-free Glycogen
        • Cold ≥ 95% Ethanol
        • 80% Ethanol

      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.

    • Advantages and Features

      Features

      • Make effective siRNAs against any gene target
      • Heterogeneous population of siRNA ensures silencing of target gene
      • From DNA template to transfection in just 1 day
      • Eliminates trial and error approach of synthetic siRNA

      Application Features

      • Gene silencing
      • Target validation

    • Related Products

      Companion Products

    • References

      1. Morlighem, J.E. et al. (2007). Biotechniques. 42, 599-606.
      2. Yang, D. et al. (2002). Proc. Natl. Acad. Sci. USA. 99, 9942-9947.
      3. Calegari, F. et al. (2002). Proc. Natl. Acad. Sci. USA. 99, 14236-14240.
      4. Donze, O. and Picard, D. (2002). Nucleic Acids Res. 30, e46.

  • Protocols, Manuals & Usage

  • Quality, Safety & Legal

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