DsiRNA and TriFECTa® RNAi Kits

Obtain potent RNA knockdown via Dicer-substrate short interfering RNAs

Dicer-substrate short interfering RNAs (DsiRNAs) are chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional, 21mer siRNAs. IDT DsiRNAs were originally developed as a collaborative effort with Dr John Rossi at the Beckman Research Institute of the City of Hope (Duarte, CA, USA). Updated design rules specific to DsiRNAs have been developed, and the resulting DsiRNAs are available only from IDT.

DsiRNAs and TriFECTa® RNAi Kits

  • Achieve sustained knockdown of cytoplasmic RNA, such as mRNA and some lncRNA, using low levels of DsiRNA (typically, 1–10 nmol)
  • Choose Predesigned DsiRNAs targeting human, mouse, or rat transcripts
    • Select from over 322,000 Predesigned DsiRNAs covering the complete transcriptomes
    • Conveniently order TriFECTa RNAi Kits (3 Predesigned DsiRNAs for the same target, 3 Control DsiRNAs, and Duplex Buffer), which are guaranteed to work*
  • Easily generate Custom DsiRNAs to sequences from any species

Controls

  • Validated positive control DsiRNAs
  • Companion qPCR primers for positive control samples
  • Validated negative control DsiRNAs

These products may not be used for human or veterinary diagnostic, prophylactic, or therapeutic purposes.

* We guarantee that at least 2 of the 3 DsiRNAs in the TriFECTa kit will produce at 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 DsiRNA indicates that >90% of the cells have been transfected, and 3) the HPRT positive control DsiRNA causes ≥90% knockdown.

DsiRNA and TriFECTa® RNAi Kits

Use our design tools to select Predesigned DsiRNAs, Custom DsiRNAs, or a TriFECTa RNAi Kit, which consists of 3 Predesigned DsiRNAs for the same target (2 nmol each in separate tubes), 3 controls (negative control plus positive controls for transfection and for a control target), and IDT Nuclease-Free Duplex Buffer.

All Predesigned DsiRNA sequences are provided after purchase.

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

Plates require a minimum of 24 DsiRNAs. Pricing for DsiRNAs in plates is per DsiRNA. Please contact custcare@idtdna.com to order DsiRNAs in plates.

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

For non-human in vivo research applications, or other applications which require larger amount of material, please contact custcare@idtdna.com for large scale ordering.

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Positive Control DsiRNAs

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Negative Control DsiRNAs

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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.

Overview of RNAi pathway

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.

Overview of DsiRNA pathway

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.

RNA interference

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IDT DsiRNAs are chemically synthesized 27mer RNA duplexes that are optimized for Dicer processing and are ideal for small scale in vitro applications. Pricing includes affinity purification, and each DsiRNA is identified by ESI mass spectrometry. All QC data is provided free of charge on the IDT website. All Predesigned DsiRNA sequences are provided after purchase.

Whenever possible, we recommend using Predesigned DsiRNAs, as these include significantly more bioinformatic analysis than is possible for DsiRNA sequences designed in real time using the custom design tool.

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). Use our online design and ordering tool to search for Predesigned DsiRNAs by gene symbol or NCBI RefSeq Accession Number.

Site selection is first performed using a proprietary algorithm that integrates both 21mer siRNA design rules, as well as updated 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. Sequences are also screened to minimize the potential for cross-hybridization and off-target effects (Smith-Waterman analysis).

Custom DsiRNAs

Use our online design and ordering tool to select DsiRNAs that target any sequence retrieved using an NCBI RefSeq Accession Number or entered as a FASTA sequence. This is ideal for working with species other than human, mouse, and rat.

Note: You may also order DsiRNAs by manually entering the sequence.

DsiRNAs in plates

If you are testing 24 or more DsiRNAs, reduce costs by ordering a multi-reaction plate of DsiRNAs (2 or 10 nmol of each DsiRNA). Contact custcare@idtdna.com.

Predesigned and Custom DsiRNAs

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Target-specific DsiRNAs

The TriFECTa Kit contains three predesigned 27mer RNA duplexes (Predesigned DsiRNAs, 2 nmol each) that are specific for a single target gene. DsiRNAs are provided in individual tubes and can be used singly or pooled, if desired.

The TriFECTa library consists of Predesigned DsiRNAs targeting the human, mouse, and rat transcriptome in the RefSeq collection. These Predesigned DsiRNAs are selected using a rational design algorithm that integrates both 21mer siRNA design rules, as well as updated 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. Sequences are also screened to minimize the potential for cross-hybridization and off-target effects (Smith-Waterman analysis).

Control DsiRNAs

It is good practice to optimize transfection conditions for each cell line studied, as well as for each type of nucleic acid used (for example, large DNA plasmids often require different transfection conditions than short dsRNA oligonucleotides). DsiRNAs can be used with all common transfection methods, such as cationic lipids, liposomes, and electroporation, depending on your cell line.

In addition to target-specific DsiRNAs, each TriFECTa kit contains three control DsiRNAs that can be used to optimize your RNAi experimental system before undertaking studies on new targets:

  • Fluorescent dye-labeled DsiRNA (Tye 563 Transfection Control DsiRNA, 1 nmol) can be used to easily monitor transfection of cultured cells
  • Positive control DsiRNA (HPRT-S1 Positive Control DsiRNA, 1 nmol) targets a site in the hypoxanthine phosphoribosyntransferase (HPRT) 1 gene that is common between human, mouse, and rat
  • Universal negative control DsiRNA (Negative Control DsiRNA, 1 nmol) does not target any sequence in the human, mouse, and rat transcriptomes

IDT Nuclease-Free Duplex Buffer

DsiRNAs are provided as RNA duplexes dried in tubes. You may use Nuclease-Free Duplex Buffer to resuspend your DsiRNAs. One 2-mL tube of Nuclease-Free Duplex Buffer is included in each TriFECTa Kit.

TriFECTa guarantee

IDT guarantees that at least 2 of the 3 DsiRNAs in the TriFECTa Kit will give at least 70% knockdown of the target mRNA when:

  1. DsiRNA is used at a 10 nM concentration and assayed by quantitative RT-PCR
  2. The fluorescent transfection control experiments indicate that >90% of the cells have been transfected
  3. The HPRT positive control DsiRNA works with the expected efficiency

TriFECTa® RNAi Kit

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Transfection efficiency control DsiRNAs

Good transfection is required for successful RNA interference using DsiRNAs. We recommend optimizing transfection conditions for each cell line studied and for each form of nucleic acid used (for example, large DNA plasmids often require different transfection conditions than shorter 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.

The dye-labeled transfection efficiency control DsiRNAs allow for rapid, easy screening of many reagents or conditions in parallel.

Endogenous positive control DsiRNAs and qPCR assays

It is possible to get good DsiRNA uptake without delivery of the oligos into the correct cytoplasmic location for effective RNAi. We recommend testing for functional knockdown using a positive control DsiRNA after checking for efficient transfection.

The HPRT-S1 Positive Control DsiRNA can be used for this purpose. With good transfection, 10 nM HPRT positive control will reduce HPRT mRNA levels by >90% at 24 hours. These controls are intended only for developing good transfection methods and knockdown is best examined at 24 or 48 hour time points. Knockdown of HPRT can slow cell growth and affect cell viability for incubation periods >72 hours. 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.

HPRT qPCR assays are available for measuring knockdown of HPRT mRNA expression in human and mouse cells.

Exogenous reporter gene control DsiRNAs

Depending on your cells, knockdown of reporter genes can be used as either positive or negative controls. For cell lines that express the EGFP or Luciferase (Firefly or Renilla) reporter gene stably or by co-transfection of an expression plasmid, DsiRNAs targeting the respective reporter gene serve as positive controls. However, for cell lines that do not express EGFP or Luciferase reporter genes, DsiRNAs  targeting the respective reporter gene can serve as negative controls. These reporter gene controls are validated, functional DsiRNAs with efficient RISC loading and, therefore, offer more control than non-targeting sequences.

Universal negative control DsiRNAs

The Negative Control DsiRNA is 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).

Control DsiRNAs (positive and negative)

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EGFP site-1 at 50 nM siRNA

A. EGFP fluorescence (compare 27+0 to 21+2 at 50 NM)

EGFP site-1 at 200 pM siRNA

B. EGFP fluorescence (compare 27+0 to 21+2 at 200 pM)

EGFP site-1 at 50 pM siRNA

C. EGFP fluorescence (compare 27+0 to 21+2 at 50 pM)

EGFP vs Duplex Concentrations

D. Dose-response (0–50 nM dsRNA)

Dose-response curve of dsRNAs transfected into NIH3T3 cells that stably express EGFP

E. Dose-response testing and cleavage of longer dsRNAs (≥27mers)

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.]

EGFP vs Days after transfection

A. Enhanced duration of RNAi by DsiRNAs

EGFP vs RNA Duplex transfected

B. DsiRNAs elicit RNAi at low concentrations

hnRNP H vs Beta actin

C. Strong knock down of protein levels by DsiRNA

La vs Beta actin

D. Potent decrease in RNA levels by DsiRNA

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.]

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

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DsiRNA transfection efficiency control

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]

Monitor DsiRNA transfection efficiency with Transfection Control DsiRNAs

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DsiRNA negative and postitive controls

Figure 4. Use a negative control DsiRNA during dose optimization and to determine your 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]

Assess RNAi function with Positive and Negative Control DsiRNAs