Get Help Sign In
ProcessingProcessing
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.

Loading...

DsiRNAs in plates

Loading...

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 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.

* With the exception of mixed base oligos, which could potentially represent multiple sequences and therefore cannot be accurately evaluated by ESI mass spectrometry.

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 nM 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]

User guides and protocols

Frequently asked questions

Certificates of analysis (COAs)

Find COAs by batch or lot number

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.

Using DsiRNA to map pain pathways in the CNS

DsiRNAs (Dicer-substrate RNAs) are chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional 21mer siRNAs. Read this example of a research group using the technology for in vivo delivery to the CNS.

The problem with pain

Chronic pain significantly impacts workplace productivity and health care costs. Once patients develop chronic pain, most will experience it for the rest of their lives. Pharmacological approaches are often unable to alleviate this condition in every patient. Unfortunately, the mechanisms behind chronic pain are poorly understood. Opiates, like morphine, are among the classic medicinal treatments, but produce many side effects and often provide poor pain control. A significant proportion of the population find little, if any, relief from opiate use. As a result, elucidating the pathways that cause pain and developing new therapeutic approaches to pain relief are important initiatives within the research community.

Targeting pain receptors

Nicolas Beaudet works with Dr Philippe Sarret at the Université de Sherbrooke (Quebec, Canada), where they study the mechanisms of pain, investigate ways to target pain receptors, and develop analgesics that circumvent problems with opiates as they are currently used in the clinic. The laboratory’s initial pharmacological studies focused on G-coupled protein receptors which are the targets for 50% of the drug market [1]. The research team wanted a more specific way to study the mechanisms than the typical pharmacological approach and so began collaborating with IDT to explore genetic strategies for treating chronic pain [2].

One of their primary targets is neurotensin. Naturally produced in the central nervous system, neurotensin is a peptide thought to play an essential role in providing analgesia. Dr Sarret’s group began working to increase the effect of endogenous neurotensin by developing a medicinal chemistry platform to synthesize more potent drugs that target neurotensin receptors. The laboratory’s focus has evolved to more precise research on the mechanisms for how neurotensin can induce analgesia using preclinical models. In proof-of-concept experiments with rats and mice, the researchers are studying the effects of several of these new molecules.

The Sarret lab is using a unique animal platform for rodents that includes behavioral devices to detect pain. While some of their new drugs are showing improved efficiency to alleviate pain in the lab, the researchers are also pursuing a genetic strategy to understand the role of neurotensin in pain modulation. Knockout mouse systems are often used for such purposes. However, the Sarret lab wanted to avoid the distorted model that often comes with knockout systems and also wanted to be able to maintain the work in rats; they opted to pursue in vivo RNA interference (RNAi) experiments. In collaboration with IDT, they used IDT Dicer-substrate RNAs (DsiRNAs) to inhibit expression of neurotensin receptors.

Two schools of thought exist for how pain is processed. Some say that pain is only processed by the brain with the spinal cord serving mainly for relaying the nociceptive stimuli and for receiving inhibition commands. Others say that the spinal cord has its own center of command, adding to the input received by the brain. The Sarret lab is focusing on the latter and is the first lab to direct in vivo RNAi specifically to the central nervous system (CNS).

DsiRNA candidate selection

The Sarret lab provided the IDs of genes encoding known or suspected receptors involved in nociception in the dorsal root ganglia of the rat spinal cord. The IDT team generated a series of DsiRNAs for these targets and tested them in vitro using cell lines expressing the genes. During screening, several assays were developed to account for variable rates of RNA degradation following target mRNA cleavage by Ago2. A minimum of 10 DisRNAs per target assay were screened to create a compound dose response curve. The IDT team found DsiRNAs yielding target knockdown of >70% at 100 pM, and IC50 values of 3050 pM. At least 2 DsiRNAs with this potency were identified per target for subsequent confirmation of in vivo biological effects, to control for potential off-target effects (OTEs). Occasionally, reagents which are very potent in vitro show reduced potency in vivo. Some of these rare cases are under study; it is suspected that gene target site sequence may differ between live rats used in the pain studies and rat cell lines used in cell culture for validation.

In vivo candidate testing

Specific delivery of DsiRNAs to the CNS involves mixing the DsiRNAs with a delivery agent (iFect, Neuromics), and injecting the mix intrathecally to directly target the spinal cord and dorsal root ganglia (Figure 1A). The researchers perform two injections at 24 and 0 hours prior to administering a pain stimulant, and then use a characterized subchronic pain scoring system to note pain level over the next 60 minutes [3]. When they inject the DsiRNA to block neurotensin signaling, they have found the animal experiences more pain (Figure 1B). This increased level of pain is a result of down regulation of the natural alleviation pathway.

dcd13_YRdsirna_fig1A
Figure 1. In vivo intrathecal delivery of neurotensin DsiRNAs block analgesic effect of neurotensin agonist. A) Confocal photomicrograph of a dorsal root ganglia section showing the uptake of Texas Red-tagged DsiRNAs by small, medium, and large neurons. Red dots correspond to DsiRNA, while the large black dots are neuron nuclei. The picture was taken 24 hr after 2 daily injections. B) Behavioral effect of 2 DsiRNAs targeting the G protein-coupled receptor Neurotensin NTS2 involved in pain modulation. A rat’s tail is immersed in a hot water bath and the latency time to flick the tail out is monitored in seconds. The baseline withdrawal latencies are under 4 sec. Injection of a NTS2-selective agonist (JMV) increases flick latency to 12 sec. If a pre-emptive intrathecal treatment of 2 daily doses of 1 μg DsiRNA is given 24 hr later, the NTS2 analgesic compound shows no more efficacy.

Beaudet and colleagues are also using DsiRNAs as an analgesic, by targeting chemokines that are believed to be involved in the CNS transmission of chronic pain. Using this model, the group targets a receptor involved in stimulating pain rather than alleviating it. A chemokine injected into the CNS will prime a severe inflammation of the spinal structures, similar to what could happen after a spinal cord trauma, for example. The injected chemokine causes immediate pain that persists for 7–10 days. When they inject DsiRNAs targeting the chemokine receptor 2 days prior to the insult, pain is inhibited for up to 7 days. By silencing the receptor via DsiRNA knockdown of its expression, pain is reduced. Beaudet says this genetic-analgesic methodology may be a new approach for situations where pharmacological approaches have proved inefficient to provide pain relief.

One of the concerns with using DsiRNAs is the potential for long-term reagent toxicity, both for the DsiRNA or the transfection reagent. The Sarret lab is able to use very low levels of DsiRNAs in these short-term delivery experiments and observe almost no side effects or off-target effects. They are now planning experiments to examine prolonged delivery of DsiRNAs. Beaudet says identifying the most appropriate transfection reagent that will not cause inflammatory responses itself has been the main concern. They have had good success with Transductin delivery reagent from IDT and have found that reagent gives them the best results so far.

The Sarret lab published the first proof-of-concept manuscript to detail in vivo work in the CNS [4], and continues to lower DsiRNA doses and examine the side effects that come with treatment. They currently have different models for some of the specific types of chronic pain (e.g., neuropathic, arthritis, and cancer) and are trying to accurately represent what is happening in the clinic to humans. The next step for the lab will be to implant osmotic pumps to deliver DsiRNAs to the intrathecal space for sustained release over 28 days.

An eye toward the clinic

These experiments move researchers closer to providing clinicians with solutions for alleviating long-term chronic pain. Patients who experience intractable pain (such as cancer pain that persists until death) may be the best candidates to try new procedures. In the worst-case scenarios, like bone cancer, patients may be offered invasive spinal therapies with the latest innovative treatments, such as DsiRNA injection directly into the spinal space. Beaudet imagines injections targeting receptors or channels involved in exacerbating painful influx may reset the neurons that are firing at a high rate and contributing to the chronic pain. Alternatively, it may be possible to install an intrathecal pump to deliver the DsiRNA analgesic over a longer period of time. As with the animal studies, clinical success will partially depend on finding a transfection reagent appropriate for human trials with no inflammatory side effects. Beaudet is optimistic, but cautions that it is much too early to say if this approach will be successful therapeutically.

Research profile

Nicolas Beaudet joined Dr Philippe Sarret’s team at the Faculty of Medicine of the Université de Sherbrooke (Quebec, Canada) 6 years ago. Prior to that, he studied biotechnology at the Université de Sherbrooke and Birmingham Cancer Institutes (UK). He further patented a nanoencapsulation technology for cancer therapy which earned him a master’s degree in applied microbiology at the Armand-Frappier Institute of the INRS University (Canada). His previous work focused on gene therapy and formulation. In the Sarret lab, Beaudet has collaborated with IDT as well as the Quebec Pain Research Network to develop new devices that evaluate chronic pain and on new drugs to sooth intractable pain. In addition to his professional activities, he is currently working on his PhD in health economy with a focus on evaluating the direct costs of low back pain. Nicolas Beaudet (3rd from right) and Dr Sarret (2nd from right) are pictured with Sarret lab staff and graduate students.

References

  1. Sarret P, Doré-Savard L, et al. (2010) Using RNA Interference to Downregulate G Protein-Coupled Receptors. In: Stevens C. (eds) Methods for the Discovery and Characterization of G Protein-Coupled Receptors. Neuromethods, vol 60. Humana Press.
  2. Sarret P, Doré-Savard., et al. (2010) Application of Dicer-Substrate siRNA in Pain Research. In: Erdmann V., Barciszewski J. (eds) RNA Technologies and Their Applications. RNA Technologies. Springer, Berlin, Heidelberg.
  3. Sarret P, Doré-Savard L, and Beaudet N. (2010) Direct application of siRNA for in vivo pain research. Methods Mol Biol, 623: 383−395.
  4. Doré-Savard L, Roussy G, et al. (2008) Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy, 16(7): 1331–1339.

Published Sep 12, 2011
Revised/updated Jun 2, 2014