By performing several RNAi screens with siRNA libraries, Ambion scientists identified five procedures that significantly improved siRNA library screen results.
- Minimize cytotoxicity
- Reduce siRNA concentration
- Use individual, validated siRNAs, not siRNA pools
- Correct for cell number
- Note cell type differences
Here, we share our recommendations for achieving RNAi screening success. These guidelines will give your data more validity.
(1) Minimize Cytotoxicity
(2) Reduce siRNA Concentration
Figure 1. siRNA Concentration vs. mRNA Knockdown. HeLa cells were transfected with 1-100 nM of six different Silencer™ Pre-designed siRNAs (Ambion). siRNAs transfected at 1-3 nM concentrations often elicited effective silencing.
Figure 2. Low siRNA Concentrations are Effective in Functional Assays. HeLa cells were transfected with siRNA to nine different targets. One day post-transfection, cells were treated with etoposide to induce apoptosis. Three days post-transfection, cells were lysed and monitored for caspase 3 activity. Results for each well are graphed relative to the average caspase 3 activity from a negative control transfected sample.
(3) Use Individual, Validated siRNAs, Not siRNA Pools
Figure 3. siRNA Pools vs. Single siRNAs. The expression levels of 59 kinases were reduced in individual HeLa cell populations in 96 well plates by transfecting, in triplicate, three single siRNAs (Panel A; #1, #2, #3) as well as a pool of siRNAs (Panel B; #1+#2+#3) targeting each kinase. After assaying cell number with Alamar Blue™ (Biosource) caspase 3 assays were performed as described for Figure 2. Yellow arrows indicate results that were confirmed targets (i.e. inhibiting kinase activity via siRNA inhibits activation of the apoptosis pathway as measured by caspase 3 activity) by both methods. Red arrows indicate false negative results from pooled transfections, and green arrows indicate false positive results from pooled transfections. The graph (Panel C) shows representative examples of genes identified as positive results, false negative from pooled assays, false positive from pooled assays, and negative results. The yellow line indicates the 70% threshold used to determine "hits."
The transfected cells were monitored for their ability to activate caspase 3 following induction of apoptosis. "Hits" were defined as cells that had at least 30% less caspase 3 activity than negative control transfected cells. Interestingly, the hits identified by the pools of siRNAs only partially overlapped with the hits identified by the three individually transfected siRNAs (Figure 3A).
Since our definition of a validated hit is a gene for which two siRNAs yield the same phenotype in a screen, we were able to use the results of the screen to determine if the pool results could be validated with the individual siRNA results. Indeed, only 60% of the siRNA pool hits could be validated with the results of the individual siRNAs. Non-validated hits were considered false positives. The pools gave a false positive rate of 40%.
Of greater concern was that six of the twelve genes for which two or more single siRNAs yielded a hit, failed to register as hits in the siRNA pool screen. The 50% false negative rate observed for pools indicates that screening with siRNA pools can result in lost opportunities for target gene identification. A sampling of confirmed positives, false positives, false negatives, and confirmed negatives is shown in Figure 3B.
We have measured the false positive and false negative rates associated with screening using a single siRNA for each gene target or pools of three siRNAs. The siRNA pool versus individual siRNA screen analysis was repeated using nine different phenotypic assays including cell proliferation, cell morphology, and cytoskeleton formation assays. The pools consistently produced false positive rates of greater than 50% and false negative rates that exceed 40% (data not shown). As seen in Figure 4, the false positive and false negative rates associated with using a single siRNA per target gene (#1, #2, #3) are lower than when siRNA were pooled. To minimize the number of False Negative (missed) genes and the number of False Positive (not confirmed by other specific siRNAs) genes, researchers should analyze the combined results from separate transfections of three different gene-specific siRNAs (#1+#2+#3). For larger screening experiments where this may not be feasible, results from single siRNA transfections of one (#1, #2, #3) or two (#1+#2, #1+#3, #2+#3) gene-specific siRNA still reduced the number of missed or False Positive genes compared to transfection of pools of siRNAs.
Figure 4. Screening With Individual siRNAs Results in Fewer False Positives and False Negatives Than Screening With Pooled siRNAs. Inhibition of 11 of 59 kinase genes via transfection of siRNAs inhibited etoposide-activated apoptosis (monitored by caspase 3 activity, see Figure 3). By definition, repression of Confirmed Positive genes resulted in low caspase 3 activity in assays from at least two gene-specific siRNAs. To examine the accuracy of screening methods for identifying important genes, we analyzed the results of transfection experiments involving either a mixture of three siRNAs that target the same gene (siRNA pool) or one of the three siRNAs (#1, #2, #3).
(4) Correct for Cell Number
Figure 5. Cell Number Differences in Cells Transfected with Distinct siRNAs. Panel A. HeLa cells (5,000 per well in 96-well plates) were transfected using siPORT™ NeoFX™ (Ambion) in triplicate with three different siRNAs targeting each of 59 different kinase-specific siRNAs. Three days post-transfection, cells were fixed and counted using the ArrayScan® V (Cellomics). The average number of cells per view field for wells transfected with the various gene-specific siRNAs were plotted. An approximately four-fold variability in cell number in wells transfected with different siRNAs suggests that results from well-based assays (e.g. ELISA, enzymatic activity assay, reporter signal measurements) must be normalized by the number of cells in each well. Panel B. HeLa cells were transfected as described in Panel A. Caspase 3 activity was monitored as described in Figure 3. Green arrows note genes that fell below the selection threshold irrespective of whether normalization was used; yellow arrows note genes that fell below only when normalized by cell number; and red notes the gene that shifted above the selection threshold when normalized to cell number.
(5) Note Cell Type Differences
Figure 6. Response to Specific siRNA-mediated Gene Inhibition Varies with Cell Type. HeLa (A) and HepG2 (B) cells were transfected in triplicate with three siRNAs targeting 13 different genes or a scrambled siRNA negative control. Cells were counted three days after transfection. Although MAPK13 (brown) inhibition decreases cell proliferation in both cell types, CDK7 (red) and GAPDH (blue) inhibition preferentially decreases cell proliferation in HeLa cells, and MAPK12 (yellow) preferentially decreases cell proliferation in HepG2 cells.