DsiRNAs are 27mer duplex RNAs that demonstrate increased potency in RNA interference compared to traditional, 21mer siRNAs. Proprietary design rules produce optimized DsiRNAs that are available only from IDT.
Duplexed 27 nt RNA strands. Order as a TriFECTa Kit and receive all the necessary reagents for RNAi.
Use the DsiRNA design tool to browse our inventory of predesigned DsiRNAs, generate custom DsiRNAs, or build your own TriFECTa RNAi Kit. If you prefer to create RNA duplexes without the help of these tools, select manual entry.
DsiRNAs are chemically synthesized, 27
For ultimate convenience, you can acquire all the necessary reagents for RNA knockdown by ordering your DsiRNAs and controls in a TriFECTa RNAi Kit.
* With the exception of mixed base oligos, which could potentially represent multiple sequences and therefore cannot be accurately evaluated by ESI mass spectrometry.
Whenever possible, we recommend using predesigned DsiRNAs, as these include significantly more bioinformatics analysis than is possible for DsiRNA sequences designed in real time using the custom design tool. Sequences for all predesigned DsiRNA ordered are provided after purchase.
Over 322,000 predesigned DsiRNAs have been designed against the human, mouse, and rat transcriptomes (RefSeq Genbank collection: www.ncbi.nlm.nih.gov/RefSeq). With our online design and ordering tool, you can search for predesigned DsiRNAs by gene symbol or NCBI RefSeq accession number. Once you have selected your DsiRNA, the tool will perform automated site selection using a proprietary algorithm that integrates 21mer siRNA design rules and updated criteria specific for 27mers.
Additional analysis is performed to ensure that the chosen sites do not target alternatively spliced exons and do not include known single-nucleotide polymorphisms. Sequences are also screened to minimize the potential for cross-hybridization and off-target effects (Smith-Waterman analysis). If plan to use 24 or more DsiRNAs, you reduce costs by ordering a multi-reaction plate of DsiRNAs (2 or 10 nmol of each DsiRNA).
With the TriFECTa Kit, you receive all of the reagents you need for successful RNA knockdown. These include:
We guarantee that at least 2 of the 3 DsiRNAs in your TriFECTa Kit will give you ≥70% knockdown of your target mRNA when:
You can use our online DsiRNA tool to select DsiRNAs which target sequences in species other than human, mouse, or rat. To do so, first click “Generate Custom DsiRNA” within the tool, and then enter a NCBI RefSeq accession number or FASTA sequence.
Our DsiRNAs are compatible with all common transfection methods, including cationic lipids, liposomes, and electroporation. However, certain methods may be more efficient than others depending on your cell line. Before undertaking studies of new targets, it is best practice to optimize your RNAi experimental system with these controls.
RNA interference is a conserved pathway common to plants and mammals, where double-stranded RNAs (dsRNAs) suppress expression of genes with complementary sequences [1–2]. Long dsRNAs are degraded by the endoribonuclease Dicer into small effector molecules called siRNAs (small interfering RNAs). siRNAs are approximately 21 bases long with a central 19 bp duplex and 2‑base 3′‑overhangs. In mammals, Dicer processing occurs as a complex with the RNA-binding protein TRBP. The nascent siRNA associates with Dicer, TRBP, and Argonaut (Ago2) to form the RNA-induced silencing complex (RISC), which mediates gene silencing (Figure 1) . Once in RISC, one strand of the siRNA (the passenger strand) is degraded or discarded, while the other strand (the guide strand) remains to direct sequence specificity of the silencing complex. The Ago2 component of RISC is a ribonuclease that cleaves a target RNA under direction of the guide strand.
Although long dsRNAs (several hundred bp) are commonly employed to trigger RNAi in C. elegans or D. melanogaster, these molecules also activate the innate immune system and trigger interferon (IFN) responses in higher organisms. RNAi can be performed in mammalian cells using short RNAs, which generally do not induce IFN responses. Historically, siRNAs have been synthesized as 21mers that bypass the need for Dicer processing by directly mimic the products that are produced by Dicer in vivo.
However, it is now thought that, in addition to being a nuclease, Dicer is also required to introduce the siRNA into RISC and is involved in RISC assembly (Figure 2) [4–6]. IDT DsiRNAs are chemically synthesized 27mer RNA duplexes that are optimized for Dicer processing and show increased potency when compared with 21mer siRNAs [7–8]. Dicer-substrate RNAi methods take advantage of the link between Dicer and RISC loading that occurs when RNAs are processed by Dicer.
Figure 1. 27mer DsiRNAs (27+0) are more potent effectors of RNAi than a 21mer siRNA (21+2). Double-stranded RNA (dsRNA) names: number of duplexed bases + number of 3′ overhanging bases or – number of 5′ overhanging bases. Each graph point represents the average of 3 independent measurements. (A–D) EGFP expression levels were determined after cotransfection of HEK293 cells with a fixed amount of EGFP expression plasmid and various concentrations of dsRNAs of varying length. Transfections were performed using (A) 50 nM, (B) 200 pM, and (C) 50 pM of the indicated dsRNAs. Error bars indicate the standard deviation. (D) Dose-response testing of dsRNAs. (E) Left: Dose-response curve of longer dsRNAs transfected into NIH3T3 cells that stably express EGFP. Right: Using an in vitro Dicer cleavage assay to analyze Dicer processing of longer dsRNAs. DsiRNAs and cleavage products are shown in this 15% nondenaturing polyacrylamide gel. [Nat Biotechnol, 23(2):222–6.]
Figure 2. Enhanced duration of RNAi at lower concentrations when comparing 27mer DsiRNA (27+0) to 21mer siRNA (21+2). Double-stranded RNA (dsRNA) names: number of duplexed bases + number of 3′ overhanging bases. (A) Enhanced duration of RNAi by DsiRNAs (up to 10 days) compared to siRNA (approximately 4 days): 5 nM of DsiRNA or siRNA were transfected into NIH3T3 cells stably expressing EGFP. Duplicate samples were taken on the indicated days, and EGFP expression was determined by fluorometry. (B) DsiRNAs can elicit RNAi at low concentrations compared to siRNAs. EGFP expression was determined after dsRNAs were transfected along with the EGFP reporter construct. Target names: site-2 is EGFP-S2 and site-3 is EGFP-S3, which were both targets known to be refractory to RNAi using siRNA. (C, D) Comparison of DsiRNA and siRNA in downregulation of endogenous transcripts (that is, hnRNP H mRNA or La mRNA). (C) hnRNP H knockdown was assayed by western blot and (D) La knockdown by northern blot analyses. The dsRNAs were used at the indicated concentrations. β-Actin was used as an internal specificity and loading standard. [Nat Biotechnol 23(2):222–226.]
Figure 3. Use a Transfection Control DsiRNA to visually monitor transfection efficiency. NIH3T3 cells were transfected with the Cy® 3 Transfection Control DsiRNA. Cells were washed and examined at 24 hr after transfection. Fluorescence and phase-contrast images are overlaid. Scale bar, 100 µm. [Nat Methods 3 (2006), DOI:10.1038/NMETH919]
Figure 4. Negative control DsiRNA during dose optimization determine baseline. HeLa cells were transfected using TriFECTa DsiRNAs specific for HPRT1, SSB, STAT1, and HNRPH1 at the concentrations indicated. Relative mRNA levels were measured using qRT-PCR at 24 hr after transfection; data were normalized against an internal RPLP0 control using the Scrambled Negative Control DsiRNA (Con) as baseline (100%). [Nat Methods 3 (2006), DOI:10.1038/NMETH919]
Figure 5. The HPRT Positive Control DsiRNA delivers strong knockdown of mRNA and protein. HeLa cells were transfected with HPRT S1 Positive Control DsiRNA (10 nM) and analyzed 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. [Nat Methods 3 (2006), DOI:10.1038/NMETH919]