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HiScribe RNAi Transcription Kit |
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Prices are in US dollars and valid only for US orders.
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Description:
The HiScribe™ RNAi Transcription Kit is a complete system for the in vitro synthesis of large amounts of double-stranded RNA (dsRNA) for RNA interference (RNAi) experiments. The kit can also be used for traditional synthesis of single-stranded RNA, which can be used in numerous applications including RNA structural studies, ribozyme biochemistry, in vitro translation, RNA-protein interactions, antisense technology, and aptamer discovery (SELEX). In addition to all reagents for high-yield in vitro transcription, the kit includes the LITMUS cloning/bidirectional transcription vectors, which are ideal for all transcription applications, including both in vitro (microinjection, soaking and cell culture transfection) and in vivo (worm feeding) RNAi experiments.
RNA interference (RNAi) is a recently-discovered mechanism of post-transcriptional gene silencing in which double-stranded RNA corresponding to a gene (or coding region) of interest is introduced into an organism, resulting in degradation of the corresponding mRNA (1-3). Unlike antisense technology, the RNAi phenomenon persists for multiple cell divisions before gene expression is regained. RNAi is therefore an extremely powerful, simple method for assaying gene function. By making targeted knockouts at the RNA level by RNAi, rather than at the DNA level using conventional gene knockout technology, a vast number of genes can be assayed quickly and economically.
Recently RNAi applications have been developed for cultured insect or mammalian cells where silencing of an endogenous gene is achieved by introducing in vitro synthesized dsRNA (4,5). Processing of the synthesized RNA by hydrolysis with E. coli RNase III renders the dsRNA to the appropriate size for RNAi in most mammalian cell lines (6).
Method Overview: Synthesis of double-stranded RNA using the HiScribe RNAi Transcription Kit, both in vivo and in vitro, is accomplished by simultaneous transcription of both strands of template DNA with T7 RNA polymerase.
The HiScribe RNAi Transcription Kit contains sufficient reagents for synthesis of up to 2 mg of double-stranded RNA in 2 ml of transcription reaction. The included LITMUS™ (Logical In vitro Transcription, Multiple Unique Sites) vectors feature extensive polylinkers flanked by opposing T7 promoters. Unlike the previous generation of LITMUS vectors, the T7 promoters in the included vectors LITMUS 28i and 38i have the consensus wild-type sequence for maximum yield of transcript. LITMUS 28i and 38i are identical except for the multiple cloning sites, and feature the pUC origin for high plasmid DNA yields, the M13 origin for production of single-stranded template for sequencing or mutagenesis, and ampicillin resistance. Additionally, the polylinkers are inserted in frame to the lacZ α-fragment, allowing blue/white screening for cloned inserts by α-complementation.
For in vitro transcription, plasmid DNA containing the cloned target of interest is digested (in separate reactions) with enzymes flanking the inserts, generating a template for each RNA strand (Figure 1). Simultaneous transcription of both linearized templates will produce double-stranded RNA of a defined length, with no vector-derived sequence apart from polylinker sequence. RNA is produced in yields up to 500-1,000 µg/ml of transcription reaction (Figure 2).
Alternatively, the cloned insert is amplified by PCR using a single T7 promoter-specific primer, generating a double-stranded template whose ends are defined by the T7 promoters themselves (Figure 1). In vitro transcription of the PCR product will produce double-stranded RNA directly, without the need for separate restriction digestions. By first cloning the target sequence in LITMUS, one can amplify the sequence using a single universal T7 promoter primer, bypassing the need for long insert-specific primers containing T7 promoters which must be designed and synthesized for each target sequence.
Figure 1: Production of dsRNA from an insert cloned in LITMUS 28i.
Figure 2: In vitro production of single and double-stranded RNA using the HiScribe RNAi Transcription Kit. LITMUS 38i containing a 1.3 kb luciferase fragment was digested in separate reactions with PspOMI (P) and StuI (S), which flank the insert. The resulting linearized templates were then transcribed either separately (lanes P and S, respectively) or together (P/S) to yield the expected single-stranded and double-stranded RNA products. RNA yields were determined to be least 0.5 µg/µl by agarose gel electrophoresis.
Advantages:- High-yield (1 mg/ml)
- Double-stranded RNA produced in a single reaction
- Compatible with RNAi protocols in C. elegans, Drosophila and tissue culture
Kit Components:
2-Log DNA Ladder (0.1–10.0 kb)
High Molecular Weight (HMW) Component Mix (30X)
LITMUS 28i Control Plasmid
LITMUS 28i Vector
LITMUS 38i Vector
NTPs (10X)
Nuclease Free dH2O
RNase-Free Water
SDS-Free Gel Loading Buffer (6X)
T7 Minimal Primer (19-mer)
T7 RNA Polymerase
Transcription Buffer (10X)
Storage Conditions
Storage Temperature:
-20°C
References
- Bass, B.L. (2000) Cell, 101, 235-238.
- Fire, A. (1999) Trends Genet., 15, 358-363.
- Sharp, P.A. (2001) Genes and Devel., 15, 485-490.
- Clemens, J.C., Worby, C.A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B.A. and Dixon, J.E. (2000) Proc. Natl. Acad. Sci. USA, 97, 6499-6503.
- Billy, E. et al. (2001) Proc. Natl. Acad. Sci. USA, 98, 14428-14433.
- Yang, D. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 9942-9947.
Reagents Sold Separately
2-Log DNA Ladder (0.1–10.0 kb)
LITMUS 28i Vector
LITMUS 38i Vector
T7 Minimal Primer (19-mer)
T7 RNA Polymerase
Companion Products
dsRNA Ladder
LITMUS™ U
phi6 RNA Polymerase (RdRP)
Ribonucleotide Solution Mix
RNase Inhibitor
siRNA Marker
Legal
Licenses/Patents/Disclaimers:
Licensed Under French Application No. FR/9115710, 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 LITMUS Vectors contained within this kit may require a license from New England Biolabs.
* Use of this kit for RNA interference may require a license from the 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.
* The PCR process is covered by patents owned by Hoffman-LaRoche.
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