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ShortCut® RNAi Kit |
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Prices are in US dollars and valid only for US orders.
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Description:
The ShortCut® RNAi Kit is a complete system for the in vitro generation of short double-stranded RNA (dsRNA) mixtures, suitable for RNA Interference (RNAi) experiments in mammalian cells (Figure 1). The kit includes reagents for both high-yield in vitro transcription of large dsRNA and subsequent processing into short fragments (18-25 nucleotides) using highly concentrated RNase III with a modified reaction buffer containing manganese. The ShortCut RNAi Kit consistently produces a heterogeneous mixture of short interfering RNAs (siRNA), the sequences of which collectively span the entire target RNA (unpublished results). This method results in highly effective silencing and eliminates the need to design costly synthetic siRNAs. Each kit contains reagents for synthesis and processing of multiple dsRNAs sufficient for over 100 transfections in 24-well plate format.
Method Overview: In the ShortCut RNAi Kit Protocol, a large dsRNA is first synthesized by in vitro transcription from a DNA template. Suitable DNA templates can be generated by cloning fragments into LITMUS 28i/38i vectors (NEB #N3528, #N3538, not included) where they can be amplified using a single T7 promoter-specific primer, generating double-stranded DNA templates whose ends are defined by the T7 promoters themselves, or by PCR using specific primers with appended T7 promoters (see Additional Protocols for help in designing such primers). The supplied Biotinylated T7 Primer can be used to amplify any sequence flanked by T7 promoters. Optionally, the amplified biotinylated DNA template can be isolated by binding to Streptavidin Magnetic Beads (NEB #S1420S) and used directly as a template for transcription. In vitro transcription of the immobilized DNA template produces DNA-free double-stranded RNA.
Processing of large (typically, 150-1500 bp) dsRNA by ShortCut RNase III digestion in the presence of manganese produces a heterogeneous population of short (18-25 bp) siRNAs and completely eliminates the longer dsRNA (Figure 2). Unlike methods which use RNase III digestion (partial or complete) in magnesium buffer, ShortCut produces highly potent siRNA mixtures for RNAi in mammalian cells (Figure 3).
Figure 1: Overview of ShortCut RNAi Kit: DNA template (PCR product, linearized plasmid) is transcribed in vitro. The resulting long dsRNA is completely converted into a heterogeneous mixture of siRNA using ShortCut RNase III.
Figure 2: siRNA production by ShortCut RNase III: (A) Varying amounts of ShortCut RNase III were incubated with 2 µg of a 500 bp dsRNA for 20 minutes. (B) dsRNA fragments (1 kb and 175 bp) were digested with ShortCut RNase III. Digests were analyzed by 20% TBE polyacrylamide gel electrophoresis.
Advantages:- From DNA template to transfection in just 1 day
- Heterogenous population of siRNA ensures effective silencing of target gene
- Eliminates trial and error approach of synthetic siRNA
Kit Components:
Biotinylated T7 Primer (25-mer)
EDTA (10X)
High Molecular Weight (HMW) Component Mix (30X)
LITMUS 38iluc Control Template
MnCl2 (10X)
NTPs (10X)
RNase-free Glycogen
ShortCut Reaction Buffer (10X)
ShortCut® RNase III
siRNA Marker
T7 RNA Polymerase
Transcription Buffer (10X)
Storage Conditions
Storage Temperature:
-20°C
Figure 3: GFP Silencing in COS-7 Cells: COS-7 cells co-transfected with a plasmid expressing GFP in the absence (control) or the presence of 30 ng (4 nM) of GFP hsiRNA prepared using the ShortCut RNAi Kit. Cells were photographed 48 hours post-transfection.
References
- Fire, A. et al. (1998) Nature, 391, 806-811.
- McManus, M.T. and Sharp, P.A. (2002) Nat. Rev. Genet., 3, 737-747.
- Yang, D. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 9942-9947.
- Calegari, F. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 14236-14240.
Reagents Sold Separately
Biotinylated T7 Primer (25-mer)
ShortCut® RNase III
siRNA Marker
T7 RNA Polymerase
Companion Products
6-Tube Magnetic Separation Rack
dsRNA Ladder
HiScribe RNAi Transcription Kit
LITMUS 28i Vector
LITMUS 38i Vector
LITMUS™ U
ProtoScript® First Strand cDNA Synthesis Kit
Streptavidin Magnetic Beads
TransPass™ R1 Transfection Reagent
TransPass™ R2 Transfection Reagent
Legal
Licenses/Patents/Disclaimers:
Licensed Under French Application No. FR/9115710, U.S. Patent No. 5,795,715, EPO No. 618966B1 and Japanese Patent No. JP3515108. Use of this kit may require a license from Commissariat A L’Energie Atomique (“CEA”). For further information, please contact the Director of Life Sciences at CEA at 31-33, rue de la Fédération 75015 Paris Cedex 15 France.
Notice to Buyer/User: Non-profit buyers or users have a non-exclusive license to use this kit for RESEARCH PURPOSES ONLY*. Commercial use of the ShortCut® RNAi Kit may require a license from New England Biolabs. Use of ShortCut® RNase III for RNA interference may require a license from Carnegie Institution of Washington. For further information, contact the Director of Administration and Finance at the Carnegie Institution of Washington at 1530 P Street, N.W., Washington, DC 20005-1910. Tel: 202-939-1118.
Licensed under U.S. Patent No. 6,506,559, corresponding foreign applications/patents and U.S. Provisional Appln. No. 60/068,562.
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