Strategies for Improving siRNA-Mediated Target Knockdown
Introduction
RNAi experiments typically conclude with the detection of target knockdown via detection of mRNA, protein, or altered phenotypes to determine the extent of target knockdown. However, these results may reveal less than desirable levels of target knockdown. A common concern is that poor knockdown is due to a characteristic of the siRNA. However, as we will discuss below, with the availability of the Dharmacon SMARTselection designed siRNAs and SMARTpool reagents or pre-designed siRNA reagents with high probability of potent silencing, siRNA specificity is usually the least likely reason that poor target knockdown is observed. Here we divide the possible causes for poor target knockdown into three main categories based on extensive troubleshooting experience:
- siRNA degradation
- Poor delivery of siRNA
- Inappropriate detection methods
Experimental Issues
siRNA Degradation
Handling siRNA or any RNA product requires implementation of standard molecular biological practices to guard against loss due to degradation or reduced integrity. Factors such as the presence of ubiquitous single and double strand ribonucleases or repeated freeze/thaw cycles may lead to a reduction in the effective concentration of intact, functional siRNA.
Non-denaturing polyacrylamide gel electrophoresis (PAGE) can be used to determine the integrity of your siRNA. If the siRNA is intact, the ethidium bromide-stained gel will reveal a discrete band corresponding to the duplex. The presence of smaller sized bands, and/or a smear below the intact duplex band, or the absence of a band indicates RNA degradation by the presence of RNases and will require systematic testing of all solutions to isolate the source of RNase contamination. Depending on the sensitivity of the detection method, a discrete band of the correct size with little to no smearing suggests fully duplexed, intact siRNA (Figure 1). To avoid non-specific siRNA degradation, the following general rules should be followed:
- siRNA should be handled in an RNase-free environment
- siRNA should be re-suspended and stored as concentrated stocks of 20–100 mM
- siRNA stocks should not be exposed to greater than five freeze/ thaw cycles
- siRNA stored as a dried pellet at –20ºC is stable for 12 months
- siRNA stored as re-suspended, concentrated stocks at –20ºC is stable for 6 months
Most challenges associated with working under RNase-free conditions have been addressed with the availability of pre-designed and pre-packaged silencing reagents. By minimizing handling of these reagents, the researcher may conduct experiments without fear of siRNA degradation.
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Figure 1. Intact vs. degraded RNA. 1 µM of siRNA was exposed to RNase-free buffer or human serum and run on a 20% PAGE/TBE gel along with nucleic acid markers and stained with ethidium bromide. The intact siRNA in RNase-free buffer migrates as a distinct band corresponding to 20 bps. In contrast, the siRNA that has been exposed to serum has already started to degrade, even at “0” minutes, as evidences by the presence of bands below the intact siRNA band. After exposure to serum for 35 minutes, the siRNA has been completely degraded. The upper band corresponds to components present in human serum, while the lower band corresponds to the siRNA. |
siRNA Delivery
The inefficient delivery of siRNA into the cell whether by transfection, electroporation, or conjugation to other chemical moieties, is by far the primary reason for most observations of poor siRNA-mediated target knockdown. The ability to obtain efficient intracellular delivery will provide the best opportunity for target-specific knockdown, especially when working with rationally designed siRNAs. Preliminary experiments must be performed to find the appropriate cell density, transfection reagent, concentration of reagent, and concentration of siRNA to achieve optimal delivery with minimal toxicity. For more information on optimized transfection methods, control protocols, and recommended transfection conditions for cell lines, visit http://www.dharmacon.com, Product Information, Product Inserts.
Detection of Target Knockdown
Detection of target knockdown at the mRNA level is the most important and direct indication of siRNA-mediated silencing efficiency. There are many methods to detect mRNA including reverse transcription-PCR (RT-PCR), northern blotting, and Branched-DNA assay. Although it is not recommended, the protein concentration may be detected by Western Blotting and ELISA, or the phenotype monitored through cell viability and differentiation. However, if your detection method has poor sensitivity, you may obtain false results from your siRNA target knockdown experiments. Incorrect results may stem from poorly designed RT-PCR probes targeted against either the 5’ or 3’ end only, as opposed to bracketing the targeted region, or antibodies that lack sufficient specificity for the protein of interest. To determine if the cause for what appears to be poor target knockdown may be due to an inaccurate detection method, it is useful to assess whether both the mRNA and protein levels from the cells have consistent knockdown.
Time Course
Because the stability and half-life of mRNAs and their protein products may vary, it is important to determine the best time course for assessing target knockdown. If a prolonged experiment is performed, and the mRNA or protein levels in the cells are allowed to return to their normal level, the stage where target knockdown occurs will be missed. For example, PPARG requires a very short time course (~12 hours), as its knockdown occurs very rapidly. Conversely, it is more common that you will need a longer time course for your experiment. The experimental design should allow time for the siRNA to get into the cell, the RISC complex to reach the mRNA, and the protein present prior to knockdown to be depleted. Figure 2 demonstrates that while the VKOR siRNA may immediately knockdown the VKOR mRNA, after 10 days, the VKOR protein has yet to be reduced to the level of the VKOR mRNA. Because protein turnover rates will differ, it is important to research the literature for mRNA and protein stabilities to help determine the best time course. In addition, experimenting with the time course will minimize the possibility of poor target knockdown results.
Sequence-Specific Target Effects
Rationally designed selection algorithms, such as the Dharmacon SMARTselection siRNA design algorithm, should be used to choose siRNAs that are highly effective. Although rare, there are several sequence-specific effects that can cause poor target knockdown. These effects include splice variants, single nucleotide polymorphisms (SNPs), and siRNA target specificity. Splice variants result from alternative splicing of an mRNA transcript during the maturation process and give rise to distinct target sequences. SNPs represent a naturally occurring variation across populations. The SNP Consortium Ltd. is dedicated to documenting SNPs through a publicly available database. (http://snp.cshl.org/) The design of siRNAs should include careful review of the original target sequence by performing a BLAST analysis to determine the existence of known or unknown variants, SNPs, and to verify the specificity of the siRNA sequence. (BLAST is a search tool provided by NCBI, the National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/BLAST) However, when relying on a single siRNA, undocumented SNPs may interfere with or eliminate silencing. Thus, the Dharmacon SMARTselection designed siRNAs and SMARTpool reagents ensure that the specificity of the siRNA for its desired target is seldom the cause for poor siRNA-mediated target knockdown.
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Figure 2. Time course of Vitamin K Epoxide Reductase (VKOR) siRNA Effects on VKOR mRNA and Protein Levels in A549 cells. VKOR activity decreased continuously during this time period while its mRNA levels decreased rapidly to about 20% of normal. Reprinted with permission from authors (Li, et al. Nature. (2004) 427:541-544). |
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