siRNA Design, Manufacturing, Transfection & Detection

Introduction

RNAi (RNA interference) is a natural biological mechanism wherein siRNA (short interfering RNA) duplexes induce potent inhibition of gene expression (Figure 1). These siRNA duplexes are produced naturally when an enzyme, Dicer, cleaves long dsRNA (double-stranded RNA) into smaller fragments. The resulting 21-23 nucleotide dsRNA fragments, i.e. siRNA, then associate with an RNase-containing complex to form the RISC (RNA-induced silencing complex).

The RISC unwinds the duplex and releases the sense strand. The RISC-bound antisense strand then serves as a guide for targeting the activated complex to complementary mRNA sequences. This results in subsequent mRNA cleavage and degradation. In effect, only catalytic amounts of siRNA are required for destruction of mRNA, resulting in the knockdown or silencing of the target gene and thereby diminished protein expression. Most importantly, synthetic siRNA can also be introduced into the cell to produce the same effect.

Silencing gene expression has become an important strategy in functional genomics. Bioinformatics software, optimized siRNA reagents, and protocols have now made RNAi experiments fast and convenient. Therefore, RNAi is a useful technology for research and drug discovery.
 

Biological mechanism of RNAi. Synthetic siRNA can also be introduced into the cell to produce the same effect, making it useful for research and drug discovery

Figure 1. Biological mechanism of RNAi. Synthetic siRNA can also be introduced into the cell to produce the same effect, making it useful for research and drug discovery.
 

The siRNA Workflow

A typical siRNA experiment (Figure 2) starts with the selection of a gene target and ends in the determination of knockdown efficiency, which is interpreted with respect to the objectives of the experiment. For studies that are focused on individual gene targets, there may be a variety of decision-making steps involved, as well as process optimization requirements.

The siRNA workflow. From designs with bioinformatic tools to detection via qPCR

Figure 2. The siRNA workflow. From designs with bioinformatic tools to detection via qPCR, we support RNAi experiments from start to finish.
 

Here we will take a high-level look at some of the important factors to consider when designing and executing an RNAi experiment with siRNA. We will also describe the ways in which we have been able to overcome some of the more challenging aspects of these experiments.

Step 1: Design

The design of an siRNA may be the most important factor for a successful RNAi experiment. This is the reason that we entered into an exclusive partnership with Merck & Co. to use its proprietary Rosetta Inpharmatics design algorithm. We have used this algorithm to create MISSION® Predesigned siRNA, and we use it for Custom siRNA design requests, too.

Rosetta designs lead to enhanced performance via the following:

  • Experimental validation – The tool has been optimized based on more than three years of extensive wet-lab testing.
  • High specificity – Potential off-target effects are reduced by minimizing homology of the seed region (nucleotide residues 2-8 in the antisense strand) to the 3’ UTR (untranslated region) of non-target mRNA.
  • High potency – Predesigned and custom-designed sequences efficiently silence high and low abundance mRNA.
  • Guaranteed – Two out of three predesigned siRNA per target gene will achieve knockdown efficiencies of ≥75%.

Step 2: Manufacturing

Well-designed siRNA will not perform to expectation without high-quality manufacturing. Quality of our siRNA is assured by implementation of extensive process controls throughout the manufacturing supply chain. The most critical reagents, the phosphoramidites, are sourced internally. In addition, the sequence identity of each siRNA is verified by mass spectrometry, and to ensure that the minimum yield/quantity (for animal studies, gram quantities of material with in vivo purity are available) has been achieved, each siRNA is also checked by UV spectrophotometry. A final visual inspection ensures that the siRNA is in the correct format, correctly labeled and ready for immediate use.

Step 3: Transfection

Once an siRNA has been designed and manufactured, the next challenge is finding a way to deliver the siRNA into the cell. We have demonstrated efficient siRNA transfection and knockdown using the N-TER Nanoparticle siRNA Transfection Reagent. The reagent is a peptide that binds siRNA non-covalently, forming a nanoparticle. This nanoparticle interacts directly with lipids on the surface of the plasma membrane, thereby allowing the nanoparticle to diffuse across the membrane and deliver siRNA directly to the cytoplasm.

Other useful transfection reagents include:

Step 4: Detection

Experimentally, it is difficult to achieve complete knockdown of a gene via RNAi. While residual expression can be detected in a variety of ways, it is important to verify that the effect being detected is a result of the knockdown of the targeted gene and not an off-target effect. Detection of the knockdown can be at the transcriptional (mRNA), translational (protein), or phenotypic level.

Most mRNA and protein assays are performed 24-to-72 hours post-transfection. However, optimal time points may need to be assessed for certain target genes or experimental conditions. The recommended methods listed below are not intended to be comprehensive, but rather to provide a glimpse of the different alternatives available.

Quantification of mRNA

Detecting and measuring residual mRNA levels for the gene of interest is a direct method for monitoring knockdown efficiency. This residual mRNA may be quantified by a variety of methods, the most common of which is qPCR, either via SYBR® Green Primers or Dual-Labeled Probes.

Quantification of Protein

Some of the methods available to detect and measure protein levels include the following:

  • Western Blotting – Proteins are separated by electrophoresis and transferred to a membrane. The protein bands are then probed with labeled antibodies. We offer thousands of primary antibodies against both human and mouse proteins along with a comprehensive set of labeled anti-species secondary antibodies.
  • AQUA™ Peptides – This is a mass spectrometry-based method for the Absolute QUAntification of proteins. An AQUA Peptide™ sequence is selected from the sequence of the target protein and then synthesized with a stable isotope label, e.g. 13C and 15N. The target protein is digested enzymatically, and a known amount of the AQUA Peptide is added to the digest as an internal standard or calibrant. The masses of the native peptide and the AQUA Peptide usually differ by 8-12 Da, depending on the peptide sequence. The concentration of the target protein is then calculated based on the relative mass spectrometry signals of the target peptide and the isotope-labeled AQUA Peptide.
  • 18O Labeling Technology – This mass spectrometry-based protein assay is somewhat analogous to the AQUA strategy, except that this is designed for relative protein quantification. Proteins from two different treatment conditions are digested enzymatically and then one of the samples is labeled with 18O. The masses of the two peptides differ by 4 Da and are clearly resolved by mass spectrometry. The relative ion intensities of the two peptides are quantitative indicators of their relative abundance.
 

Conclusions

Various factors contribute to successful RNAi experiments. Design, manufacturing, transfection, and detection of siRNA are the most important elements for accurate analysis of gene silencing.

 

If additional help is needed, please consult our technical services group at oligotechserv@sial.com.

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