Determining siRNA selection

What is important when selecting an siRNA library? Design and performance are just a starting point for the use of this technology.

Large-scale siRNA screens can generate hundreds of potential ‘hits’ – genes for which one or more siRNAs give a desired phenotype. Unfortunately, in early screens with unmodified siRNA technologies and pooled siRNA approaches, many of these hits are actually because of offtarget effects.

‘The first concern is the quality of the algorithm that is used to design the siRNAs,’ says Anand Ganesan MD, PhD, assistant professor, Department of Dermatology and Biological Chemistry at the University of California, Irvine. ‘Secondly, the number of siRNAs that have been validated either by the company or as a part of other studies should be considered.’

Ambion® Silencer® Select siRNAs incorporate the latest improvements in siRNA design, off-target effect prediction algorithms and chemistry. As a result, they provide unrivalled silencing consistency, potency and specificity, and fewer failed experiments, resulting in cleaner, more consistent phenotypic data.

siRNA quality
siRNA design and structure are important; however, siRNA manufacturing quality is equally critical. Ambion® Silencer® Select siRNAs are manufactured under highly controlled processes, and are subjected to rigorous quality control procedures.

Dr Hakim Djaballah, director of the High Throughput Screening Core Facility at Memorial Sloan Kettering Cancer Center, says: ‘Cellular viability is a major concern in our screening experiments. We need to know that the contaminants that result from the production of siRNA are at extremely low levels. Often we dilute our reagents, and use them on a small scale with a small number of cells and we cannot afford for impurities to be present.’

Customisation concerns
siRNA screens are quite varied in nature. ‘Every experiment has different requirements,’ says Djaballah. ‘What is the scale of siRNA desired in each well? How are the duplexes oriented? Can the supplier provide custom aliquotting? Most importantly, what are the turnaround times for ordered libraries?’ All are critical factors in selecting a siRNA library vendor, both for the initial siRNA library purchase, as well as for hit confirmation follow-up studies.

Ambion Silencer Select siRNA libraries are available at scales of 0.1, 0.25, 1.0, 2.0 and 5.0 nmol per well. Custom aliquotting requests of all types are easily handled, and the company can supply siRNAs in your desired plate layouts to match your transfection and cell-based assay protocols.

Validation
Once hits from a library screen are identified, validation is a crucial step prior to drawing conclusions. ‘We don’t think simply using another supplier’s siRNA is a good enough method for validation if you are relying on the differences between the two suppliers’ algorithms,’ says Djaballah.

‘We prefer qRT-PCR followed by protein level measurements. We sometimes look at rescue experiments using an expression vector, but these can be tedious and there can be toxicity concerns.’

‘We validate hits by several different strategies,’ Ganesan explains. ‘We use multiple siRNAs to eliminate off targets. We carry out quantitative qRT-PCR to ensure that the siRNA impacts the expression of the target gene, and we examine the impact of these siRNAs in several different cell lines. More recently, we have also been validating our hits with shRNAs. The major cost of screens is usually not from the screen itself, but from the follow ups.’

Ambion, now within the Invitrogen family, can help clients define an appropriate follow-up and hit validation strategy. Since 2002, the company has consulted with and helped thousands of scientists using siRNAs.

Company profile

Life Technologies is a global biotechnology tools company dedicated to improving the human conditon. Its systems, consumables and services enable researchers to accelerate scientific exploration, driving to discoveries and developments that improve life.

For further information, visit: www.appliedbiosystems.com/ select.