• My NEB
  • Print
  • PDF
  • ShortCut® RNase III

    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 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.

    Highlights

    • Isolated from a recombinant source
    • Gene silencing
    • Target validation
    • Supplied with 10X Reaction Buffer

    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).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    10X EDTA500 mM
    MnCl210X
    Glycogen RNase-Free10 mg/ml
    Shortcut Reaction Buffer10X

    Advantages and Features

    Applications

    • Gene silencing
    • Target validation

    Properties and Usage

    Unit Definition

    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.

    Reaction Conditions

    1X ShortCut Reaction Buffer
    Incubate at 37°C

    Usage Concentration

    1X

    Storage Temperature

    -20°C

    Storage Conditions

    500 mM NaCl
    10 mM Tris-HCl
    0.5 mM EDTA
    1 mM DTT
    pH 8.0 @ 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.

    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. (202). Proc. Natl. Acad. Sci. USA. 99, 14236-14240.
    4. Donze, O. and Picard, D. (2002). Nucleic Acids Res. 30, e46.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What products does NEB offer for functional genomics and reverse genetics?
    1. ShortCut RNase III Digestion Protocol (M0245)
    2. Purification of siRNA by Ethanol Precipitation Protocol (M0245)
    3. Transfection Guidelines for ShortCut® RNase III (M0245)