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Oligonukleotid szintézis, szintetikus biológia

IGÉNY SZERINTI dsiRNS

 

Dicer-substrate RNAs (DsiRNAs) are chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional siRNAs and were developed as a collaborative effort between John Rossi at the Beckman Research Institute of the City of Hope and IDT. Traditional 21mer siRNAs are designed to mimic Dicer products and therefore bypass interaction with the enzyme Dicer. Dicer has been recently shown to be a component of RISC and involved with entry of the siRNA duplex into RISC. Dicer-substrate siRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway. Using this approach, sustained knockdown has been regularly achieved using sub-nanomolar concentrations. New design rules specific to DsiRNAs have been developed and are available only from IDT.

IDT also offers the TriFECTa® kit which contains three Dicer-Substrate 27mer 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.

 

DsiRNA

Dicer-substrate RNAs (DsiRNAs) are chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional siRNAs and were developed as a collaborative effort between John Rossi at the Beckman Research Institute of the City of Hope and IDT. Traditional 21mer siRNAs are designed to mimic Dicer products and therefore bypass interaction with the enzyme Dicer. Dicer has been recently shown to be a component of RISC and involved with entry of the siRNA duplex into RISC. Dicer-substrate siRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway. Using this approach, sustained knockdown has been regularly achieved using sub-nanomolar concentrations. New design rules specific to DsiRNAs have been developed and are available only from IDT.

IDT also offers the TriFECTa® kit which contains three Dicer-Substrate 27mer 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.

  • Predesigned DsiRNAs—DsiRNA RefSeq Library
  • TriFECTa® kit
  • Large-scale DsiRNAs

 

Predesigned DsiRNAs—DsiRNA RefSeq Library

Over 3,000,000 DsiRNAs have been designed against the approximately 25,000 genes from each of the human, mouse, rat, cow, dog, chicken, and chimp transcriptomes in the RefSeq Genbank collection: http://www.ncbi.nlm.nih.gov/RefSeq. Site selection is first performed using a proprietary algorithm that uses novel Dicer-substrate specific design rules. Sequences that pass this stage are next screened to minimize the potential for cross-hybridization and off-target effects (Smith-Waterman analysis) and sites that include known SNPs or alternatively spliced exons are eliminated. Finally, the local mRNA secondary structure is modeled to avoid areas with a high level of predicted structure.

 

IDT offers two types of Predesigned DsiRNA duplexes:

  • Splice common: targets all known variants of a gene in RefSeq; duplexes lie only within common extons. This constitutes the bulk of the DsiRNA collection and is the correct choice for most users. 8–10 duplexes are available for each gene target.
  • Splice specific: targets only exons present in specific splice forms. In some cases, the unique exon may be very small or comprise sequence unfavorable for selection of RNAi duplexes (please note that knock-down guarantees do not apply for these sites). This collection is intended for those researchers who have a specialized need to selectively target specific splice variants or isoforms.

 

When working with a gene target that is included in the above collection, IDT recommends using the predesigned duplexes as these include significantly more bioinformatic analysis than is possible for sequences designed in real time using the web interface design tool.

 

TriFECTa® Kit

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 sequences are from the DsiRNA library and so include options from seven genomes : human, mouse, rat, cow, dog, chicken, and chimpanzee. 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 to not target alternatively spliced exons and do not include known single-nucleotide polymorphisms. IDT guarantees that at least two of the three DsiRNA duplexes in the TriFECTa kit will give at least 70% knockdown of the target mRNA when 1) used at 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 positive control works with the expected efficiency.

 

Large-Scale DsiRNAs

The small scales are intended for use in cell culture. For in vivo use, or other applications which require larger amounts of material, please see the Large-Scale ordering product page.

 

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