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 21mer siRNAs are chemically synthesized RNA duplexes that mimic Dicer products and bypass the need for Dicer processing. Dicer-Substrate RNAs are chemically synthesized 27mer RNA duplexes that are optimized for Dicer processing and show increased potency when compared with 21mer duplexes [1, 2].
The TriFECTa® Kit contains three Dicer-substrate 27mer 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 27mer 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 21mer siRNA design rules as well as new 27mer-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.
- Kim DH, et al. (2005) Synthetic dsRNA Dicer-substrates enhance RNAi potency and efficacy. Nat Biotechnol, 23(2):222–226.
- Rose SD, et al. (2005) Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res, 33(13):4140–4156.