More and more researchers are using small interfering RNAs (siRNAs) to reduce the expression of specific mammalian genes. siRNAs are short, double stranded RNA molecules that can target mRNA of a specific (complementary) sequence for degradation via a cellular process known as the RNA interference (RNAi) pathway. Applications of siRNAs include functional genomics, drug target validation, and cell signaling pathway analysis, to name just a few.

Currently, there are five methods for generating siRNAs for gene silencing studies:

1. Chemical synthesis

2. In vitro transcription

3. Digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, E. coli, RNase III)

4. Expression in cells from an siRNA expression plasmid or viral vector

5. Expression in cells from a PCR-derived siRNA expression cassette

The first three methods involve in vitro preparation of siRNA that is then introduced directly into mammalian cells by transfection, electroporation, or by another method. The last two methods rely on the transfection of DNA-based vectors and cassettes that express siRNAs within the cells. Each of these methods has its advantages and drawbacks. The best method for generating siRNAs will depend on the goals of the experiment. This article briefly describes the five methods, presents their advantages and disadvantages, and discusses the types of applications for which they are best suited. Figure 1 summarizes this information.

*Depends on purification/deprotection options selected and format (e.g., annealed and ready to transfect versus single strands supplied lyophilized)

siRNA Design
All of the above methods, except #3, require the design of individual siRNA sequences prior to siRNA preparation. Although more is being learned about the design parameters that influence siRNA effectiveness, it remains an inexact science. In general, Ambion scientists find that it is best to design and test 3–4 siRNA sequences for each targeted gene, and then use the one that most effectively induces RNAi in subsequent experiments. For the latest recommendations on siRNA design and a free web-based siRNA design tool, see the following page.

In Vitro Preparation
Method #1: Chemical Synthesis
Although more expensive than any of the other methods, chemically synthesized siRNAs require almost no work by the researcher to produce. Ambion and several other companies provide high quality, chemically synthesized siRNAs on a custom basis. One of the major benefits of chemical synthesis is the large yield of high purity siRNA obtained. Drawbacks include the price and turnaround times (typically 4–12 days depending on synthesis and purification options). Because of the relatively high price tag to screen the recommended four siRNA sequences per target, many researchers find it beneficial to screen siRNA sequences using a less expensive preparation method, such as in vitro transcription, and then have the most effective sequence(s) synthesized chemically.

Best for:
Studies that require large amounts of a defined siRNA sequence

Not suitable for:
Screening siRNA sequences (cost prohibitive), long term studies

Method #2: In Vitro Transcription
siRNAs can be readily prepared by in vitro transcription. siRNAs produced by this method are considerably less expensive than their chemically synthesized counterparts, making them a more cost-effective choice for screening siRNA sequences. In addition, they can be produced more quickly than chemically synthesized siRNAs. Ambion's Silencer™ siRNA Construction Kit makes the preparation of siRNAs by in vitro transcription an easy, straightforward process. Once template deoxynucleotides are obtained, the procedure takes about 24 hours, with little hands on time. Disadvantages of this method include the limited scale up potential (although each reaction produces enough siRNA for hundreds of transfections) and the fact that it requires more hands-on time from the researcher compared to chemically synthesized siRNAs which are simply purchased. It should be noted that in vitro transcribed siRNAs work as well as chemically synthesized siRNAs and usually at lower concentrations–0.5-20 nM vs. 50-100 nM concentration per transfection; Figure 2).

Best for:
Screening siRNA sequences or when the price of chemical siRNA synthesis is an obstacle

Not suitable for:
Long term studies or studies that require large amounts of a single siRNA sequence

Figure 2. 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.

Method #3: Digestion of Long dsRNA by an RNase III Family Enzyme
One of the major drawbacks of all the other methods of siRNA production is the need to design and test several siRNA sequences before an effective one can be identified. Preparation of siRNA cocktails overcomes this limitation. In this method, long dsRNA is prepared by in vitro transcription using a template that typically encodes a 200–1000 nt region of the target mRNA. The dsRNA is then digested in vitro with RNase III (or Dicer) to produce a population, or cocktail, of siRNAs. After a purification step to remove any undigested dsRNA, the siRNA cocktail is ready to be transfected into cells in a manner similar to an individual siRNA. Because many different siRNAs are included in the cocktail, efficient gene knockdown is virtually guaranteed.

The major benefit of the dsRNA digestion approach is the ability to bypass the testing steps involved in selecting an effective siRNA sequence, saving researchers both time and money (note that RNase III reactions are typically quite a bit cheaper than those performed with Dicer). One drawback of this approach, however, is the theoretical potential for nonspecific silencing effects, particularly for closely related genes. Most research to date indicates that this does not pose a problem (1-4). For researchers interested in using this method, Ambion provides the Silencer siRNA Cocktail Kit (RNase III), which includes everything needed to prepare siRNA cocktails (except the transcription template). Ambion scientists have used the Silencer siRNA Cocktail Kit to knockdown the expression of many different genes, including c-fos, GAPDH, La, ß-actin, and Ku-70. A representative of one of these experiments is depicted in Figure 3.

Best for:
Fast and inexpensive analysis of loss of function phenotypes

Not suited for:
Long term studies or studies that require a single, defined siRNA sequence

Figure 3. Gene Silencing with the Silencer™ siRNA Cocktail Kit. A population of siRNAs targeting 200 nt of the Ku-70 mRNA was prepared with the Silencer siRNA Cocktail Kit (RNase III) and transfected into HeLa cells at a final concentration of 100 nM. Cells were analyzed 48 hours later by immunofluorescence. Ku-70 levels were reduced 86% in cells transfected with the siRNA cocktail, compared to non-transfected controls.

In Vivo Expression
All of the methods described so far rely on the in vitro preparation of siRNAs. The use of siRNA expression vectors and PCR-based expression cassettes, however, relies on in vivo transcription of siRNAs from DNA templates introduced into cells. A major advantage of these two approaches is that there is no need to work directly with RNA.

Method #4: siRNA Expression Vectors
Most siRNA expression vectors rely on one of three different RNA polymerase III (pol III) promoters to drive the expression of a small hairpin siRNA in mammalian cells (1–4). These promoters include the well-characterized human and mouse U6 promoters and the human H1 promoter. RNA pol III was chosen to drive siRNA expression because it naturally expresses relatively large amounts of small RNAs in mammalian cells and it terminates transcription upon incorporating a string of 3–6 uridines. To use these vectors, two oligodeoxynucleotides encoding the desired short hairpin RNA sequence are ordered, annealed, and cloned into the vector downstream of the pol III promoter. Because cloning is involved, the procedure takes several days, and sequencing the region containing the insert is required. However, this limitation is balanced by the ability to produce large quantities of vector once the vector is shown to work well in gene silencing experiments.

Without a question, the main advantage of siRNA expression vectors is that they are the only method that is amenable to long term studies. Vectors with antibiotic resistance markers can be used to reduce the expression of targeted genes for several weeks or longer. Transient selection of cells transfected with selectable marker containing plasmids also permits the enrichment of cells that have taken up the plasmid. This can help compensate for low transfection efficiencies in difficult to transfect cells. Recently, several groups including Ambion have begun preparing retroviral and other viral vectors for siRNA expression. These offer the added advantage that cells can be transduced for gene silencing studies, reducing problems associated with inefficient plasmid transfection.

Ambion currently offers ten different pSilencer™ siRNA Expression Vectors. Plasmids are available with the mouse U6, human U6 or human H1 pol III promoter. The latter two promoters are also available in vectors containing puromycin, neomycin, or hygromycin resistance genes. Silencing of GFP by pSilencer 2.1-U6 hygro is shown in Figure 4.

Best for:
Long term and other studies in which antibiotic selection of siRNA containing cells is desired

Not suitable for:
Screening siRNA sequences (note: screening siRNA sequences is possible, but is time and labor intensive with vectors)

Figure 4. 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.

Method #5: siRNA Expression Cassettes
siRNA expression cassettes (SECs) are PCR-derived siRNA expression templates that can be introduced into cells directly — without first being cloned into a vector. Initially described by Castanotto and colleagues (5), SECs include an RNA pol III promoter upstream of a sequence encoding an siRNA hairpin followed by an RNA pol III termination site. In contrast to siRNA expression vectors, which require cloning and sequencing prior to use and thus take 1–2 weeks to prepare, SECs can be generated by PCR in less than a day. SECs thus provide an excellent screening tool to find the most effective siRNA sequence, or to identify the best combination of promoter and siRNA in the experimental system being used. In fact, SECs provide the perfect complement to siRNA expression vectors. By incorporating restriction sites at their ends, SECs found to effectively elicit gene silencing can be readily cloned into a plasmid or viral vector to create an siRNA expression vector. The siRNA expression vector can then be used for stable expression and long term studies. One disadvantage of SECs is that they are not as easily transfected into cells as siRNAs. As new transfection agents and protocols are developed, however, SEC transfection ease and efficiency should increase. Because they are generated by PCR, SECs are not amenable to scale up without being cloned into plasmids.

For preparation of SECs and subsequent cloning into vectors with selectable markers, Ambion provides the Silencer™ Express siRNA Expression Cassette Kits. Silencer Express Kits are available with either a mouse U6, human U6 or human H1 promoter element. Figure 5 shows data from an experiment in which SECs containing three different promoters were tested for their ability to drive siRNA expression and induce knockdown of c-fos expression.

Best for:
Screening siRNA sequences and testing promoters before preparing vectors
Not suitable for:
Long term studies until cloned into vectors containing selectable markers

Figure 5. Variable Reduction in Target Gene Expression Using SECs with Different Promoters. siRNA Expression Cassettes featuring the mouse U6 (Mo-U6), human U6 (Hu-U6), and human H1 (Hu-H1) promoters and encoding a c-fos-specific hairpin siRNA were transfected into HeLa cells. 72 hours post-transfection, the cells were assessed using nuclear staining with DAPI and immunofluorescence using a c-FOS antibody. Non-transfected cells (NT) as well as cells transfected with an SEC expressing a negative control siRNA (scramble) demonstrate wild-type levels of c-FOS. The relative level of reduction in gene expression was quantified and is provided in the bar graph.

Summing It Up
The use of RNAi to induce gene silencing is an exciting new method for creating loss of function phenotypes. Figure 1 summarizes the five different methods for producing siRNAs. For more information about each of these methods, including protocols, visit the RNAi Resource at www.ambion.com/RNAi.