siRNA Screening: Development of Hit Stratification Strategies

Did your initial round of RNAi screening produce some hits? How do you determine which hits are true positives?

Now that you have identified some positive hits on your initial round of RNAi screening, you need to take the next step – hit stratification. Which hits are real? Which are most important? Which ones should you follow up? How do I figure this all out?

This poster compares different approaches to hit stratification and validation after an initial screen. For this step, standard siRNA reagents (siGenome) deconvoluted from a pooled set of four were compared to a pooled set of four specificity enhanced (ON-TARGETplus) reagents. High confidence hits were similar, but to more closely explore the validity of low confidence hits, a chimeric approach was used whereby a gene-specific seed sequence is introduced into a non-targeting siRNA scaffold to rule out false negatives. These seed-matched controls revealed seed-mediated off-target events. Taken together, this work resulted in proposed hit stratification strategies, particularly useful for sorting out lower confidence hits.

Dharmacon scientists participate in numerous scientific conferences every year. We make select posters and presentations available from these activities because of their scientific and educational value.