Gene Silencing

Planning and Executing siRNA Experiments—Good Practices for Optimal Results

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Custom 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. For more information, please visit the TriFECTa page.

Product Configuration

  • The DsiRNA pricing above is for unmodified duplexes only. RNA duplexes can be ordered through the IDT RNA ordering module as ssRNAs or as dsRNA with a variety of modifications.
  • For in vivo use, or other applications which require larger amount of material, please inquire with Customer Care for large scale ordering. 
ProductPricing
2 nmole Screening DsiRNA Duplex$95.00 USD
2 nmole Screening DsiRNA Plate Duplex$75.00 USD
10 nmole Screening DsiRNA Duplex$145.00 USD
10 nmole Screening DsiRNA Plate Duplex$115.00 USD
40 nmole Screening DsiRNA Duplex$225.00 USD

DsiRNA

In cells, small interfering RNAs (siRNAs) are produced by enzymatic cleavage of long dsRNAs by the endoribonuclease Dicer. The siRNAs associate with the RNA Induced Silencing Complex (RISC) in a process that is facilitated by Dicer. Dicer-Substrate RNAi methods take advantage of the link between Dicer and RISC loading that occurs when RNAs are processed by Dicer. Traditional 21mer siRNAs are chemically synthesized RNA duplexes that mimic Dicer products and bypass the need for Dicer processing. In contrast, our Dicer-substrate RNAs (DsiRNAs) are chemically synthesized 27mer RNA duplexes that are optimized for Dicer processing and loading into the RISC complex. This results in increased potency when compared to traditional 21mer duplexes.The DsiRNA duplexes are ideal for small scale in vitro applications. Pricing includes affinity purification and each duplex is identified by ESI mass spectrometry. All QC data is provided free of charge on the IDT website.

  • 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. The TriFECTa product page is available here

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.

Figure 1

A)

EGFP site-1 at 50 nM siRNA

B)

EGFP site-1 at 200 pM siRNA

C)

EGFP site-1 at 50 pM siRNA

D)

EGFP vs Duplex Concentrations

E)

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

Figure 1. DsiRNAs are More Potent Effectors of RNAi Than a 21+2 siRNA. EGFP expression levels were determined after cotransfection of HEK293 cells with a fixed amount of EGFP expression plasmid and varying concentrations of dsRNAs. (A–C) Transfections were performed using (A) 50 nM, (B) 200 pM and (C) 50 pM of the indicated dsRNAs. (D) Dose-response testing of longer dsRNAs. Transfections were performed with the indicated concentrations of dsRNA. Right, depicts in vitro Dicer reactions with the same longer RNAs. Concentrations and conditions were as described in Methods. (E) Dose-response curve of dsRNAs transfected into NIH3T3 cells that stably express EGFP. Each graph point represents the average (with s.d.) of three independent measurements. (Nat Biotechnol, 23(2):222–6.)

Figure 2

A)

EGFP vs Days after transfection

B)

EGFP vs RNA Duplex transfected

C)

hnRNP H vs Beta actin

D)

La vs Beta actin

Figure 2. Features of DsiRNA in RNAi. (A) Enhanced duration of RNAi by DsiRNAs. Levels of EGFP were determined after transfection of 5 nM of a 21+2 siRNA or the DsiRNA into NIH3T3 cells stably expressing EGFP. Graphic representation of EGFP silencing mediated by a 21+2 siRNA as compared to the DsiRNA. Duplicate samples were taken on the indicated days and EGFP expression was determined by fluorometry. (B) DsiRNAs can elicit RNAi. The dsRNAs were transfected along with the EGFP reporter construct, and EGFP expression was determined (Methods). Column 1, mock; column 2, 21+2 siRNA targeting EGFPS2; column 3, DsiRNA targeting EGFPS2; column 4, 21+2 siRNA targeting EGFPS3; column 5, DsiRNA targeting EGFPS3. (C, D) Comparison of 21mer siRNA and DsiRNA in downregulation of endogenous transcripts. RNAs for a 21+2 siRNA and DsiRNA were designed to target sites in the hnRNP H mRNA (C) or La mRNA (D). HnRNP H knockdown was assayed by western blot and 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 in both experiments. (Nat Biotechnol 23(2):222–226.)

Validated Control DsiRNA Duplexes and other Control Reagents

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A successful RNAi experiment starts with good transfection. It is good practice to optimize transfection conditions for each cell line studied as well as for each form of nucleic acid employed (for example, large DNA plasmids often require different transfection conditions than short dsRNA oligos). It may be necessary to empirically test a number of different cationic lipids (or other approaches) to establish a protocol that performs optimally with each cell line employed. Use of a dye-labeled transfection control oligo allows for rapid, easy screening of many reagents in parallel.

When optimizing transfection methods, IDT recommends using dye-labeled oligos at 10 nM (or less); higher concentrations can increase the amount of non-specific binding which can cause background and falsely elevate the apparent success of transfection.

The following dye-labeled RNA duplexes are available as ReadyMade­™ products for this purpose. RNA oligos can be labeled with other dyes as a custom order.

DescriptionPricing
1 nm TEX 615™ DS Transfection Control$78.00 USD
5 nm TEX 615™ DS Transfection Control$195.00 USD
1 nm TYE™ 563 DS Transfection Control$78.00 USD
5 nm TYE™ 563 DS Transfection Control$195.00 USD

Fluorescent Transfection Efficiency Control Duplexes

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Use of dye-labeled control oligos is not sufficient by itself to optimize transfection. It is possible to get seemingly good dye-oligo uptake without delivery of the oligos into the correct cytoplasmic location for functional RNAi. Transfection conditions that "pass" the dye-labeled study should also be tested for functional knockdown using a positive control siRNA.

The "HPRT-S1 DS Positive Control" duplex can be used for this purpose. When transfection is good, theHPRT mRNA will reduce HPRT mRNA levels by >90% at 24 hours when used at 10 nM. Please note that the HPRT control is intended only to develop good transfection methods and 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 duplex can be employed in human, mouse, rat, and Chinese hamster (CHO) cells. Other genomes may require customized controls.


DescriptionPricing
1 nm HPRT-S1 DS Positive Duplex Control$60.00 USD
1 nm HPRT-Ss DS Positive Duplex Control$60.00 USD
1 nm HPRT-Bt DS Positive Duplex Control$60.00 USD
5 nm HPRT-S1 DS Positive Duplex Control$150.00 USD
5 nm HPRT-Bt DS Positive Duplex Control$150.00 USD

IDT recommends studying functional transfection efficiency by examining mRNA levels 24 hours after transfection. Alternatively, western blots can be performed at 48–72 hours.


DescriptionPart #Pricing
1 nm Human HPRT Sybr™Green Primer Set51-01-08-04$25.00 USD
1 nm Mouse HPRT Sybr™Green Primer Set51-01-13-07$25.00 USD
5 nm Human HPRT Sybr™Green Primer Set51-01-08-05$60.00 USD
5 nm Mouse HPRT Sybr™Green Primer Set51-01-13-08$60.00 USD

Endogenous Gene Positive Control Duplexes and Primers

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When qRT-PCR is performed it is necessary to have an internal standard to control for RNA loading. While many different housekeeping genes have been used for this purpose (such as β-Actin, GAPDH, or Cyclophilin), most of these genes show fluctuation in expression levels with different treatments and are not as invariant as is needed for a true internal qRT-PCR control. IDT has developed the following SYBR® Green assays suitable for use an internal normalization standard in qRT-PCR analysis (ΔΔCt method). When combined with use of the cloned copy number control plasmids, these reagents permit relative RNA mass-loading normalization plus absolute quantitative PCR to be performed.


DescriptionPricing
1 nm Human RPLP0 Sybr™Green Primer Set$25.00 USD
5 nm Human RPLP0 Sybr™Green Primer Set$60.00 USD

Internal Control Primers for qRT-PCR Analysis

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Reporter genes can be used both as positive controls and as negative controls and so are very useful reagents. If your cell line expresses the reporter either stably or via co-transfection of an expression plasmid, the anti-EGFP or anti-Luciferase (Firefly or Renilla) DsiRNAs can function as a positive control. If your cell line does not express these reporter genes, then the anti-EGFP or anti-FLuc DsiRNAs can function as negative controls. Importantly, these DsiRNAs are validated, functional duplexes with known efficient RISC loading and therefore offer an added level of control that non-targeting sequences cannot offer.


DescriptionPricing
1 nm EGFP-S1 DS Positive Control$60.00 USD
5 nm EGFP-S1 DS Positive Control$150.00 USD
1 nm FLuc-S1 DS Positive Control$60.00 USD
5 nm FLuc-S1 DS Positive Control$150.00 USD
1 nm RLuc DS Positive Control$60.00 USD
5 nm RLuc DS Positive Control$150.00 USD

Exogenous Reporter Gene Positive Controls

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Negative control duplexes have been developed which do not target any sequence in the human, mouse, or rat transcriptomes. They can be employed as a universal negative controls for DsiRNA transfections. These are non-targeting sequences. IDT recommends the NCI negative control over the DS scrambled negative. The EGFP and FLuc DsiRNAs may also be used as negative controls if functional, targeting duplexes are desired (see Exogenous Reporter Gene Positive Controls above).


DescriptionPricing
1 nm Negative Control (DS NC1)$60.00 USD
5 nm Negative Control (DS NC1)$150.00 USD
1 nm Negative Control (DS ScrambledNeg)$60.00 USD
5 nm Negative Control (DS ScrambledNeg)$150.00 USD

Universal Negative Control