Small Interfering RNAs (siRNAs) are short double-stranded RNA molecules that can target and degrade complementary mRNAs via a cellular process termed RNA interference (RNAi). Alternate methods for generating siRNA are: (i) vector based in vivo expression, (ii) chemical synthesis, (iii) in vitro transcription (IVT), (iv) RNAse III mediated hydrolysis and (v) PCR based siRNA expression cassettes. We have evaluated these diverse methodologies and found them to offer varying advantages over one another.
Figure 1. Use of Chemically Synthesized and in Vitro Transcribed siRNAs to Induce Gene Silencing. siRNAs targeting ß-Actin were prepared by chemical synthesis (Ambion) or by in vitro transcription using Ambion's Silencer™ siRNA Construction Kit. HeLa cells were plated at 30,000 cells per well in a 24 well tissue culture plate containing glass slides. The cells were transfected 24 hours after plating, using 2 µl siPORT™ Lipid (Ambion) according to the manufacturer's protocol, at a final siRNA concentration of 75 nM. Immunofluorescence analysis was performed 96 hr post transfection using mouse anti-Human ß-Actin primary antibody and a FITC conjugated anti-mouse IgG secondary antibody. Photographs were taken using the appropriate fluorescent filters and quantified using MetaMorph software. Note that both siRNA preparation methods resulted in >_ 95% reduction in ß-actin protein levels.
Figure 2. Schematic of a Typical siRNA Expression Vector. A. Vector Diagram. B. siRNA encoding insert. C. Hairpin siRNA.
Figure 3. Long Term Silencing of GFP with pSilencer™ 2.1-U6 hygro. HeLa cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6 hygro containing an insert encoding an siRNA targeting cycle 3 GFP or pSilencer 2.1-U6 hygro without an siRNA-encoding insert. Following transfection, the cells were selected with hygromycin. Three weeks following selection, the cells were analyzed for GFP expression by fluorescence microscopy. Green: GFP. Blue: DAPI stained nuclei. GFP levels were remarkably reduced (94%) in cells transfected with the GFP siRNA-encoding pSilencer 2.1-U6 hygro siRNA Expression Vector as compared to those transfected with an "empty" siRNA expression vector.
Figure 4. siRNA Expression Plasmids Under Transient Selection to Induce Gene Silencing. HeLa cells were plated at 60,000 cells per well and then co-transfected, using 1.2 µl FuGENE 6 (Roche) according to the manufacturer's protocol, with 400 ng GFP-expressing plasmid and 400 ng of each of 6 different siRNA expressing plasmids containing a selectable marker (Ambion) and encoding an siRNA targeting GFP. The cells were placed under selection at 24 hours post transfection using 300 µg/ml Hygromycin, 2.5 µg/ml Puromycin, or 2000 µg/ml G418. At 48 hours post transfection, the antibiotic containing media was removed and replaced with normal growth media. At 72 hours post transfection, the cells were harvested and analyzed. Blue: DAPI stained nuclei. Green: GFP.
Figure 5. Silencing of Gene Expression by PCR-based siRNA Expression Cassettes. A. Schematic representation of the PCR strategy used to yield the PCR-based siRNA expression cassettes [Castanotto et al., (2002) RNA 8:1454]. B. Inhibition of exogeneous EGFP expression using siRNA-encoding PCR cassettes. HeLa cells were co-transfected with PCR cassettes encoding an EGFP or scrambled control siRNA and a target EGFP expressing plasmid and analyzed by fluorescence microscopy. High levels of EGFP expression can be detected in control cells, but not in cells transfected with EGFP siRNA-encoding PCR products. C. Inhibition of endogeneous human GAPDH expression using siRNA-encoding PCR cassettes. PCR cassettes encoding a human GAPDH and a scrambled control siRNA were transfected into HeLa, DU145, and HEK 293 cells and analyzed by Northern Blot. GAPDH expression levels were reduced by >80% in cells treated with GAPDH siRNA-encoding PCR cassettes over control cells.
Figure 6. Comparison of siRNAs Prepared by Chemical Synthesis vs RNase III Digestion of Long dsRNA. A population of siRNAs targeting GAPDH was prepared by synthesizing and purifiying a 200 bp dsRNA, corresponding to the first 200 bases downstream of the AUG start site of GAPDH mRNA, with the MEGAscript RNAi Kit (Ambion) and then cleaving 15 µg of the transcript with 15 U RNase III (Ambion; Panel A). HeLa cells were plated at 30,000 cells per well in a 24 well tissue culture plate on glass cover slips. The cells were transfected 24 hours later with 100 nM, 50 nM, 25 nM, or 12.5 nM final concentration of the siRNA population or a chemically synthesized siRNA known to efficiently silence GAPDH. The cells were harvested after 48 hours and the reduction in protein levels was examined by immunofluorescence microscopy. The level of GAPDH in the cells was reduced efficiently by siRNAs prepared by both chemical synthesis and RNase III digestion.
| Chemical synthesis | RNase III digestion of dsRNA | siRNA Expression Vector | PCR Expression Cassette | In vitro transcription | |
|---|---|---|---|---|---|
| Requirements | 2 21-mer RNA oligos | Transcription template (200-800 bp region flanked by T7 promoters) | 2 55-60-mer DNA oligos | 2 ~55-mer DNA oligos | 2 29-mer DNA oligos |
| Turnaround time (total preparation/synthesis time) | 4 days to 2 weeks* | 1 day + transcription template preparation time | 5+ days + DNA oligo | ~ 6 hours + DNA oligo | 24 hours + DNA oligo |
| Hands on time | Little to none* | Moderate | High | Moderate | Moderate |
| Testing to find optimal siRNA sequence | Required | Not needed | Required | Required | Required |
| Ability to label siRNA (i.e., for analyzing siRNA uptake or localization by fluorescence microscopy) | No | No | Yes | ||
| Relative ease of transfection | Good | Fair | Good | Good | |
| Selectability (i.e, antibiotic selection) | No | ||||
| Useful for long term studies | No | No | Yes, with selection | No | No |
| Ability to scale up synthesis | Yes | Limited | Yes | Limited | Limited |
| Monitor transfection efficiency of entire population | No | No | Yes | No | No |
| Relative cost per gene (not including labor) | High | Low | Moderate | Moderate | Moderate |
*Depends on purification/deprotection options selected and format (e.g., annealed and ready to transfect versus single strands supplied lyophilized)
Summary
- Permits long-term and stable gene silencing
- Possibility to use inducible/repressible markers
- Use of viral vectors
Chemical and IVT synthesis:
- Allows rapid screening of multiple targets
- High siRNA homogeneity and purity
RNase III mediated hydrolysis:
- Eliminates the need for screening of target site
- Overcomes variability in silencing by synthetic/IVT siRNA
- Cost-effective for functional genomic studies
PCR based siRNA expression cassettes:
- Ideal for screening siRNA sequences prior to cloning in a vector
- Rapid and inexpensive procedure