functional_genomics

Dicer-Substrate Short Interfering RNAs (DsiRNAs) and TriFECTa® Kits

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.

  • Achieve sustained knockdown of cytoplasmic RNA using low amounts of DsiRNA
  • Select from over 320,000 predesigned DsiRNAs or easily generate your own
  • Conveniently obtain all of the knockdown reagents you need by ordering DsiRNAs in a TriFECTa RNAi Kit

Ordering

DsiRNAs and TriFECTa Kits in tubes

Duplexed 27 nt RNA strands. Order as a TriFECTa Kit and receive all the necessary reagents for RNAi.

ProductPricing
DsiRNA, 2 nmol$95.00 USD
DsiRNA, 10 nmol$145.00 USD
TriFECTa® RNAi Kit$395.00 USD

DsiRNAs in plates

ProductPricing
DsiRNA in plates, 2 nmol$75.00 USD
DsiRNA in plates, 10 nmol$115.00 USD

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.

For non-human in vivo research applications or other applications that require larger amounts of material, please email custcare@idtdna.com.

DsiRNAs are chemically synthesized, 27 nt RNA duplexes that are optimized for Dicer processing and are ideal for small-scale in vitro applications. These 27mer duplexes have increased potency in RNAi compared to traditional 21mer siRNAs. DsiRNAs were originally developed as a collaborative effort with Dr John Rossi of the Beckman Research Institute of the City of Hope (Duarte, CA, USA). Updated design rules that have been developed at IDT have resulted in potent DsiRNAs that are available only from IDT. Each DsiRNA is purified and identified by ESI mass spectrometry. Unless otherwise noted, DsiRNAs are provided dry in tubes. All QC data is provided free of charge on our website.

For ultimate convenience, you can acquire all the necessary reagents for RNA knockdown by ordering your DsiRNAs and controls in a TriFECTa RNAi Kit.

Predesigned, custom, and control DsiRNAs

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.

Predesigned DsiRNAs

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).

TriFECTa RNAi Kit

With the TriFECTa Kit, you receive all of the reagents you need for successful RNA knockdown. These include:

  • 3 Predesigned DsiRNAs that are specific for a single target gene
  • 3 control DsiRNAs for optimizing your RNAi experimental setup:
    • TYE 563 Transfection Control DsiRNA, 1 nmol
    • HPRT-S1 Positive Control DsiRNA, 1 nmol
    • Negative Control DsiRNA, 1 nmol
  • Nuclease-Free Duplex Buffer (2 mL) for resuspending your DsiRNAs, which are delivered dry

TriFECTa Kit guarantee

We guarantee that at least 2 of the 3 DsiRNAs in your TriFECTa Kit will give you ≥70% knockdown of your target mRNA when:

  1. The DsiRNA is used at 10 nmol concentration and assayed by qPCR
  2. Fluorescent transfection control experiments indicate >90% of cells have been transfected
  3. The HPRT positive control DsiRNA works with the expected efficiency

Custom DsiRNAs

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.

Control DsiRNAs

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.

  • Transfection efficiency controls (Cy®3, TEX 615, and TYE 563-labeled DsiRNAs): Dye-labeled, transfection efficiency control DsiRNAs allow for rapid, easy screening of many reagents or conditions in parallel. We recommend optimizing transfection conditions for each cell line studied and for each form of nucleic acid used (large DNA plasmids, for example, often require different transfection conditions than short DsiRNAs). It may also be necessary to empirically test different transfection reagents (or other approaches) to establish a protocol that performs optimally with each cell line used.
  • Endogenous gene positive controls and qPCR assays (HPRT-S1 DsiRNAs and qPCR assays): It is possible to get good DsiRNA uptake without delivering your oligos to the correct cytoplasmic location for effective RNAi. We recommend testing for functional knockdown using a positive control DsiRNA after checking for efficient transfection.
    With good transfection, 10 nmol HPRT-S1 positive control DsiRNA will reduce HPRT mRNA levels by >90% after 24 hours. Since knockdown of HPRT can slow cell growth and affect cell viability for incubation periods >72 hours, it is important to examine your cells at 24 or 48 hr timepoints. Due to sequence similarity, the HPRT-S1 control DsiRNA can be used in human, mouse, rat, and Chinese hamster (CHO) cells. Other genomes may require custom controls. To confirm functional performance, use HPRT qPCR assays to measure HPRT mRNA expression levels in human and mouse cells.
  • Exogenous reporter gene controls (DsiRNAs against EGFP or Luciferase): Depending on your cell line, knockdown of reporter genes can be used as either positive or negative controls. For cell lines that express EGFP or Luciferase reporter gene (either stably or by co-transfection of an expression plasmid), DsiRNAs targeting the respective reporter gene will serve as positive controls. However, for cell lines that do not express EGFP or Luciferase reporter genes, DsiRNAs targeting the respective gene will serve as negative controls. Due to their efficient RISC loading, using validated DsiRNAs as functional reporter gene controls offer more control than non-targeting sequences.
  • Universal negative controls (non-targeting and scrambled DsiRNAs): The Negative Control DsiRNA is a non-targeting DsiRNA that will not interact with any sequences in the human, mouse, or rat transcriptomes. If making a choice, we recommend using the Negative Control DsiRNA, instead of the Scrambled Negative Control DsiRNA. For cells that do not express the respective reporter gene, the EGFP and luciferase DsiRNAs may be used as negative controls if functional, targeting DsiRNA are desired (see Exogenous reporter gene positive controls, above).

RNA interference

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) [3]. 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.

Figure 1. The RNA-induced silencing complex (RISC) pathway in mammalian cells. In laboratory experiments, siRNA, similar to the guide strand, interact with RISC.

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 2. Mechanism for DsiRNA function in the RISC pathway.

References

  1. Hannon GJ, Rossi JJ. (2004) Unlocking the potential of the human genome with RNA interference. Nature, 431:371–378.
  2. Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature, 431:343–349.
  3. Chendrimada TP, Gregory RI, et al. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature, 436:740–744.
  4. Lee YS, Nakahara K, et al. (2004) Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell, 117:69–81.
  5. Pham JW, Pelllino JL, et al. (2004) A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell, 117:83–94.
  6. Tomari YC, Matranga C, et al. (2004) A protein sensor for siRNA asymmetry. Science, 306:1377–1380.
  7. Kim DH, Behlke MA, et al. (2005) Synthetic dsRNA Dicer-substrates enhance RNAi potency and efficacy. Nat Biotechnol, 23(2):222–226.
  8. Rose SD, Kim DH, et al. (2005) Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res, 33(13):4140–4156.

27 nt DsiRNAs are more potent effectors of RNAi than 21 nt siRNAs

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.]

Monitor DsiRNA transfection efficiency with Transfection Control DsiRNAs

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]

Assess RNAi function with Positive and Negative Control DsiRNAs

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]

GMP and OEM

If you require oligos that are approved for use in molecular diagnostic applications, or if you are interested in our OEM services, please click here.