TriFECTa® RNAi Kit (Pre-designed DsiRNA Kit)
TriFECTa® kit 은 Genbank에 있는 human, mouse, rat의 RefSeq collection으로 부터 미리 디자인 한 duplex 세트에서 선택한 세 개의 Dicer-Substrate 27-mer duplexes 로 구성되어 있으며 이들 duplex는 특정 유전자를 target으로 합니다. TriFECTa® 의 27-mer sites는 기존의 21 mer siRNA 디자인 룰과 새로운 27 mer 디자인 기준을 병합한 알고리즘에 의해서 선택됩니다. 또한 선택된 사이트가 alternatively spliced exon을 target으로 하거나 알려진 SNP 사이트를 포함하지 않도록 분석이 행해지는 등 몇 단계에 걸쳐서 최적화 됩니다.
이러한 세 개의 target-specific duplexes 외에 TriFECTa®kit에는 RNAi실험에 필요한 세 개의 control이 포함됩니다.
Transfection control : Cy3TM -labeled transfection control RNA duplex 또는 Cal Flour® Red 610-labeled transfection control
Negative control : a scrambled universal negative control RNA duplex (DS Scrambled Neg)
Positive control : Dicer-Substrate RNA duplex (HPRT-S1 DS Positive Control) -human, mouse, and rat 에 공통으로 존재하는 HPRT
(hypoxanthine guanine phosphoribosyltransferase 1) gene 을 target으로 하며 10 nM농도로 사용했을 때 90 % 이상의 knockdown이 나타남.
IDT는 TriFECTa® kit에 있는 세 개의 Dicer-Substrate duplexes 중 한 개가 10 nM concentration으로 사용 됐을 때 target mRNA를 적어도 70% knockdown 시키는 것을 보장합니다.
단 fluorescent transfection control duplex 가 90 % 이상의 세포에서 나타나고 HPRT positive control 이 작동하는 것을 qPCR로 실험 했을 때 보장해 드립니다. Knock down 실험은 mRNA를 target으로 하는 실험이기 때문에 Western Blot 데이터는 인정되지 않습니다.
TriFECTa® Kit 구성 및 가격: 소비자가 530,000원
(특별가 : 2013년 6월 까지 적용 금액)
••Three target-specific Dicer-Substrate siRNA duplexes (2 nmoles each)
••Fluorescent-labeled transfection control duplex: TYE563
••HPRT-S1 DS Positive Control duplex (1 nmole)
••DS Scrambled-Neg, universal negative control duplex (1 nmole)
••RNase Free Duplex Buffer (100 mM KAc/30 mM HEPES pH 7.5)
TriFECTa® kit 디자인은 아래 디자인 툴을 통해서 직접 확인 할 수 있습니다.
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.
Synthetic dsRNA Dicer-substrates enhance RNAi potency and efficacy. Kim, D.H., et al., Nat Biotechnol, 23(2): 222-6 (2005).
Functional polarity is introduced by Dicer processing of short substrate RNAs. Rose, S.D., et al., Nucleic Acids Res, 33 (13): 4140-56 (2005).