Functional Genomics Lab Expertise

The Functional Genomics Lab experts at NCATS have in-depth experience working with siRNA and RNAi screening technology to evaluate gene expression. These scientists routinely perform the following types of activities in support of NIH investigator-driven screening projects.

Common Seed Analysis to Prioritize Hits and On- or Off-Target Effects

A common source of error in RNAi experiments is when RNAi turns off genes other than the intended targets. These “off-target effects” can be detected by comparing the experimental results of an RNAi to all other similar RNAi, enabling scientists to determine if the effect observed is specific to the gene being targeted or if it occurs with all similar RNAi.

Seed Hexamer graph

In this plot, red triangles indicate the experimental results from siRNAs that turn off the SIAH3 gene. Black circles are plotted for all similar RNAi and demonstrate clearly that the results for SIAH3 are separated from these and are not an error caused by off-target effects.

Testing Multiple siRNAs to Increase Confidence in Genes with Consensus

Because many genes are identified in error in a typical RNAi screen, follow-up experiments using different RNAi are an important way that scientists can gain confidence in their results.

A graph displays the number of small interfering RNAs (siRNAs) against each of 10 different genes that are active in an assay for parkin translocation. Number of active siRNAs is divided into three different groups: the primary screen, the follow-up screen and the sum of the two screens. The overall graph conveys how testing with multiple siRNAs increases confidence that the identified genes are actually involved in the pathway of interest.

In this bar plot, the number of active siRNAs identified for each gene is displayed for the primary (initial screen) and follow-up experiments.

Separation of False Positives from True Positives Using C911 Controls

In addition to common seed analysis, RNAi experts at NCATS have developed a novel technology to validate identified genes: C911 controls. C911 siRNAs are modified siRNAs that retain off-target effects but eliminate the normal function of the siRNA.

A bar graph displays small interfering RNA (siRNA) experimental results for four genes that confirm with C911 controls and five genes that do not confirm with C911 controls. For genes that confirm, the bars are shorter for the C911 controls, indicating that the phenotype is lost when knockdown is lost. For genes that did not confirm, the bars are the same length, indicating that loss of knockdown does not influence the phenotype, so the original effect must be off-target.

By comparing experiments using an siRNA to experiments using the same siRNA with the C911 modification, scientists can tell the difference between true (green plot) and false (red plot) positive results.