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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
  • Useful for dsRNA removal from IVT samples – See optimization recommendations in our application note
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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 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,
    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

      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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