Design and test two to four siRNA sequences per gene.
To find a potential target site, scan the length of the gene of interest for AA sequences. Record the AA and the 3' 19 nucleotides as potential siRNA target sites. Potential target sites should then be evaluated by a BLAST analysis against the GENBANK database to disqualify any target sequence with significant homology to other genes. When possible, siRNAs should be designed to regions of target mRNA with low secondary structure. Ambion has a siRNA Target Finder and Design Tool at www.ambion.com/techlib/misc/siRNA_finder.html to simplify this procedure.

Choose siRNAs that have a low GC content.
Ambion researchers find that siRNAs with 40­55% G/C content are more active than those with a G/C content higher than 55%.

Purify in vitro transcribed siRNA.
Ensure size and purity of all siRNAs to be transfected. For the highest level of purity we recommend using glass fiber filter binding and elution or gel purification using a 15­20% acrylamide gel to remove excess nucleotides, short oligomers, proteins and salts in the reactions. Note: Chemically synthesized RNAs usually require purification by gel electrophoresis.

Avoid RNases!
Trace amounts of ribonucleases can sabotage siRNA experiments. Since RNases are present throughout the laboratory environment ­ on your skin, in the air, on anything touched by bare hands or on anything left open to the air ­ it is important to take steps to prevent and eliminate RNase contamination. Ambion offers a complete line of products designed to detect and eliminate RNases.

Maintain healthy cell cultures and strict protocols for good transfection reproducibility.
In general, healthy cells are transfected at higher efficiency than poorly maintained cells. Subculturing cells routinely at a low passage number ensures that there will be minimal instability in continuous cell lines from one experiment to the next. When performing optimization experiments we recommend transfecting cells within 50 passages. After 50 passages cells should be utilized since transfection efficiency drops over time.

Avoid antibiotic use.
Ambion recommends avoiding the use of antibiotics during plating and up to 72 hours after transfection. Antibiotics have been shown to accumulate to toxic levels in permeabilized cells. Some cells and transfection reagents require serum-free conditions for optimal siRNA delivery. We suggest a pilot transfection experiment in both normal growth media and serum-free media to determine which condition is best for each transfection.

Transfect siRNAs using optimized reagents.
Use an optimized siRNA transfection reagent and protocol for your cell type. The choice of transfection reagent is critical for success in siRNA experiments. It is essential to use transfection reagents formulated to deliver small RNAs (most commercially available transfection reagents were designed for large plasmid DNA, not small RNA molecules). Also, some reagents have been developed for the transfection of specific cell lines while others have broader specificity. Ambion's Silencer™ siRNA Transfection Kit provides reagents specifically designed to transfect siRNAs into eukaryotic cells.

Use an appropriate positive control to optimize transfection and assay conditions.
For most cells, housekeeping genes are suitable for positive controls. Transfect target cells with several concentrations of an siRNA specific to your chosen positive control. (This also applies to your experimental target siRNA.) Measure the reduction in the control protein or mRNA level compared to untransfected cells 48 hours after transfection. Too much siRNA can lead to cell toxicity and death. Ambion offers positive control siRNAs against a variety of gene targets.

Use a negative control siRNA to distinguish nonspecific effects.
An appropriate negative control can be designed by scrambling the nucleotide sequence of the most active siRNA. Be sure to do a homology search to ensure that it lacks homology to the genome of the organism being studied.

Use labeled siRNAs for protocol optimization.
Fluorescently labeled siRNA can be used to analyze siRNA stability and transfection efficiency. Labeled siRNA is also useful for study of siRNA subcellular localization and for use in double label experiments (with a labeled antibody) to track cells that receive siRNA during transfection and to correlate transfection with down-regulation of the target protein.

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