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TriFECTa® RNAi Kit

The TriFECTa kit contains three Dicer-Substrate 27-mer duplexes, targeting a specific gene, that are selected from a predesigned set of duplexes from the RefSeq collection of human, mouse, and rat genes in Genbank.

Please visit the Dicer-Substrate siRNAs page for more information on individual custom products.

TriFECTa® RNAi Kit

$395.00 USD

Kit Contents:

  • Three target-specific Dicer-Substrate siRNA duplexes (2 nmoles each)
  • Fluorescently-labeled transfection control duplex: TYE 563™ (1 nmole)
  • HPRT-S1 DS Positive Control duplex (1 nmole)
  • NC1, negative control duplex (1 nmole)
  • RNase Free Duplex Buffer (100mM KAc/30 mM HEPES pH 7.5)

These products are not for use in humans or non-human animals and may not be used for human or veterinary diagnostic, prophylactic, or therapeutic purposes.

IDT guarantees that at least two of the three Dicer-Substrate duplexes in the TriFECTa kit will give a least 70% knockdown of the target mRNA when 1) used at a 10 nM concentration and assayed by quantitative RT-PCR, 2) the fluorescent transfection control duplex indicates that >90% of the cells have been transfected, and 3) the HPRT positive control works with the expected efficiency.

In cells, small interfering RNAs (siRNAs) are produced by enzymatic cleavage of long dsRNAs by the RNase-III class endoribonuclease Dicer. The siRNAs associate with the RNA Induced Silencing Complex (RISC) in a process that is facilitated by Dicer. Dicer-Substrate RNAi methods take advantage of the link between Dicer and RISC loading that occurs when RNAs are processed by Dicer. Traditional 21-mer siRNAs are chemically synthesized RNA duplexes that mimic Dicer products and bypass the need for Dicer processing. Dicer-Substrate RNAs are chemically synthesized 27-mer RNA duplexes that are optimized for Dicer processing and show increased potency when compared with 21-mer duplexes [1, 2].

A Schematic of the Initial Steps in the RNAi Pathway After Introduction of dsRNA into a Cell. A. Dicer-substrate 27mers are bound and cleaved by Dicer, then passed into the RISC assembly in a sequence-specific orientation. B. Synthetic siRNAs (19-22mers) are bound by Dicer, and passed to RISC without any specific orientation.

The TriFECTa® kit from IDT contains three Dicer-substrate 27-mer RNA duplexes that are specific for a single target gene. Duplexes are provided in individual tubes and can be used singly or pooled, if desired. The TriFECTa library consists of predesigned 27-mer Dicer substrate siRNAs from seven of the genomes in the RefSeq collection, including human, mouse, and rat. The gene sequences are based upon the RefSeq database in Gen Bank (http://www.ncbi.nlm.nih.gov/RefSeq/). TriFECTa duplexes are selected using a rational design algorithm that integrates both traditional 21-mer siRNA design rules as well as new 27-mer-specific criteria. Additionally, analysis is performed to ensure that the chosen sites do not target alternatively spliced exons and do not include known single-nucleotide polymorphisms. The TriFECTa library can be accessed online.

In addition to target-specific duplexes, each TriFECTa kit contains three controls: a fluorescent dye-labeled duplex (Tye 3 DS transfection control), a ‘universal’ negative control duplex (NC1) that targets a site that is absent from human, mouse and rat genomes, and a positive control duplex (HPRT-S1 DS positive control) that targets a site in the hypoxanthine phosphoribosyntransferase (HPRT) 1 gene that is common between human, mouse, and rat. These control reagents can be used to optimize the RNAi experimental system before undertaking studies on new targets. It is good practice to optimize transfection conditions for each different cell line studied as well as for each different form of nucleic acid used (for example, large DNA plasmids often require different transfection conditions than short dsRNA oligonucleotides). Dicer-substrate RNA duplexes can be used with all commonly used transfection methods, such as cationic lipids, liposomes, and electroporation.

IDT guarantees that at least two of the three Dicer-Substrate duplexes in the TriFECTa kit will give a least 70% knockdown of the target mRNA when 1) used at a 10 nM concentration and assayed by quantitative RT-PCR, 2) the fluorescent transfection control duplex indicates that >90% of the cells have been transfected, and 3) the HPRT positive control works with the expected efficiency.

References

  1. Synthetic dsRNA Dicer-substrates enhance RNAi potency and efficacy. Kim, D.H., et al., Nat Biotechnol, 23(2): 222-6 (2005).
  2. Functional polarity is introduced by Dicer processing of short substrate RNAs. Rose, S.D., et al., Nucleic Acids Res, 33 (13): 4140-56 (2005).

Figure 1

 

 

Figure 1. Transfection Controls and Dose Optimization. (a) NIH3T3 cells were transfected with the TriFECTa Cy3 transfection control duplex. Cells were washed and examined at 24h after transfection. Fluorescence and phase-contrast images are overlaid. Scale bar, 100 µm. (b) HeLa cells were transfected using TriFECTa duplexes specific for HPRT1, SSB, STAT1, and HNRPH1 at the concentrations indicated. Relative mRNA levels were measured using qRT-PCR at 24 h post-transfection; data were normalized against an internal RPLP0 control using the ‘DS scrambled neg’ duplex as baseline (100%). Con, control. (Nature Methods 3 (2006), Application Note published online)

Figure 2

Figure 2. HPRT mRNA and Protein Knockdown. HeLa cells were transfected with HPRT-S1 duplex at 10 nM and collected at the indicated time points. (a) HPRT mRNA amounts were measured by qRT-PCR. (b) HPRT protein levels were assessed by western blot; Β-actin loading standard is shown. Each lane represents a separate transfection. (c) HPRT protein levels were averaged and relative knockdown at the indicated times after transfection was quantified. (Nature Methods 3 (2006), Application Note published online)