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Frequently Asked Questions: siRNA Screening Service

1. What are the advantages of siRNA screening?
2. What is the screening process of the siRNA library?
3. How do you analyze the data?
4. Are siRNA screens reliable and reproducible?

1. What are the advantages of siRNA screening?

Validating novel targets or a broad range of targets is challenged by the lack of known small molecule inhibitors for each target. The use of RNA interference utilizing siRNAs provides a special solution to this problem. siRNA screening addresses a wider number of targets and achieves specificity by target-specific knockdown. Adding siRNA screening to the early drug discovery screening programs can help to identify targets that traditional biochemical and cell-based screening may miss.

2. What is the screening process of the siRNA library?

We usually use a customer’s list of siRNAs desired for testing to develop a screening protocol that is identical for all cells being tested. The screening protocol utilizes a number of different reagents, and in advanced techniques is programmed into a robot for the process to be completed in an orderly manner. Results are composed of data relating to the expression of certain, pre-determined genes. In the case that a segment of siRNA is effective at limiting expression, the resulting data will show a significant decrease in that gene’s expression. For a number of drug-development applications, this data can show any off-target effects and demonstrate the on-target specificity and effectiveness of a novel therapeutic or drug.

3. How do you analyze the data?

A lot of work has gone into identifying appropriate approaches to analyze RNAi data to compensate for assay bias and enrich for true hits by statistical and bioinformatics analysis. There is likely a limit to what statistics and analysis can provide given the degree of biological variation and pragmatically. Therefore, it could be best to be more generous with thresholds for defining hits and spend more time on further experiments.

4. Are siRNA screens reliable and reproducible?

The similarity between replicates within any one screen is usually high, that is to say, screens are self-consistent. But the frequency with which primary hits can be reproduced in secondary screens is variable. For any cell, the phenotype resulting from a reduction in the level of a target protein could depend on:

1) The rate and extent of the reduction;
2) Any specific phenotypes elicited by the transfection process per se;
3) The degree of functional redundancy of the protein and whether the protein is involved in various cellular processes;
4) Any contributing phenotypic effects of off-target actions of the RNAi reagent.

Due to the possibility for variability in the phenotypes elicited by individual siRNAs, it is improbable that any one screen will give a complete inventory of all of the activities involved in a biological process in either across all cell lines or one cell line. Therefore, such data is probably best garnered by multiple screens possibly across a number of lines or with distinct complementary readouts.

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