Guaranteed knockdown of RNA in loss-of-function studies

The RNA interference (RNAi) pathway is a conserved, gene-regulation mechanism found in most eukaryotes that normally influences development and protects the genome from viruses and transposable elements. However, the RNAi pathway can be coopted for therapeutic and research use. After extensive studies to find effective delivery methods, potential RNAi therapies are in clinical trials (e.g., for blood disorders, some cancers, and liver diseases).

RNAi also has become the experimental tool of choice for studying the effects of gene knockdown, especially when targeting RNA in the cytoplasm. In mammalian systems, small interfering RNAs (siRNAs) are typically transfected into cells or injected into animals. The siRNA enters the RNAi pathway through incorporation into the RNA-induced silencing complex (RISC), which also includes Dicer, TRBP, and Argonaut 2 proteins [1, for overview or RNAi].

Why use DsiRNAs?

We recommend transfection or electroporation of Dicer-substrate siRNA (DsiRNA) to obtain sustained RNA knockdown in cultured cells. These 27mer duplex DsiRNAs are more potent than traditional 21mer siRNAs [2,3] and can be used at low levels (typically 1–10 nmol) to minimize off-target effects. Predesigned DsiRNAs targeting human, mouse, and rat transcriptomes, are selected using a rational design algorithm that integrates 21mer siRNA design rules and 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 (SNPs). Sequences are also screened to minimize the potential for cross-hybridization and off-target effects (Smith-Waterman analysis).

A cost-effective kit for Predesigned DsiRNA and controls

Whether you are new to RNAi or an experienced user, the TriFECTa RNAi Kit for human, mouse, or rat targets is a convenient, economical option for obtaining Predesigned DsiRNAs and important control DsiRNAs necessary for successful experiments. (Note: if you are studying a species other than human, mouse, or rat, use our Custom DsiRNAs design and ordering tool to choose your DsiRNAs.)

The TriFECTa Kits contain the following:

1. 3 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.

2. 3 control DsiRNAs that can be used to optimize your RNAi experimental system before beginning new target studies:

• Fluorescent dye–labeled DsiRNA (Tye 563 Transfection Control DsiRNA, 1 nmol) can be used to easily monitor transfection of cultured cells (Figure 1A)
• 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 (Figure 1B)
• Universal negative control DsiRNA (Negative Control DsiRNA, 1 nmol) does not target any sequence in the human, mouse, and rat transcriptomes

3. Nuclease-Free Duplex Buffer (2 mL tube) is included in each TriFECTa Kit. DsiRNAs are provided as RNA duplexes, delivered dry in tubes. Use Nuclease-Free Duplex Buffer to resuspend your DsiRNAs.

Figure 1. Visualization of transfection efficiency and mRNA knockdown using DsiRNA Controls. (A) High transfection efficiency of DsiRNA into HeLa cells. Overlay of fluorescence and phase-contrast images is shown. HeLa cells were transfected at a final concentration of 10 nM with the TYE™ 563 Transfection Control DsiRNA using X-tremeGENE^® transfection reagent (Roche). Cells were imaged 24 hours post transfection. (B) mRNA knockdown measured by RT-qPCR using low levels of DsiRNA. HeLa cells were plated at a density of 2 x 10⁴ cells/well in a 24 well plate. 24 hours later, the cells were transfected with HPRT-S1 Positive Control DsiRNA or negative control DsiRNA using Oligofectamine™ transfection reagent. 24 hours post transfection, RNA was isolated (SV96 Total RNA Isolation Kit, Promega), and HPRT levels were measured by RT-qPCR using an ABI 7000 real-time PCR machine (Thermo Fisher Scientific). Relative expression was normalized to RPLP0 internal control mRNA using the negative control as baseline (100%).

TriFECTa guarantee

Use the Predesigned DsiRNA ordering tool to select the DsiRNAs that will be included in your TriFECTa kit. We guarantee that at least 2 of the 3 Predesigned DsiRNAs in the TriFECTa Kit will result in at least 70% knockdown of the target mRNA when:

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

Visit www.idtdna.com/DsiRNA for more information about DsiRNA and DsiRNA controls.