As discussed previously, deconvoluted Dharmacon siGENOME pools often give surprising results. (Deconvolution is the process of testing the 4 siRNAs in a pool individually. This is usually done in the validation phase of siRNA screens.)
One way to compare the relative contribution of target gene and off-target effects is to calculate the correlation between reagents having the same target gene or the same seed sequence. One of the first things we do when analysing single siRNA screens is to calculate a robust form of the intraclass correlation (rICC, see discussion at bottom for more about this).
Recently we were analysing deconvolution data from Adamson et al. (2012) and calculated the following rICC’s. (The phenotype measured was relative homologous recombination.)
Grouping variable rICC 95% confidence interval Target gene 0.040 -0.021-0.099 Antisense 7mer 0.383 0.357-0.413 Sense 7mer 0.093 0.054-0.129
Besides the order of magnitude difference between target gene and antisense seed correlation (which is commonly observed in RNAi screens), what stands out is the ~2-fold difference between the correlation by target gene and sense seed.
Very little of the the sense strand should be loaded into RISC, if the siRNAs were designed with appropriate thermodynamic considerations (the 5′ antisense end should be less stable than the 5′ sense end, to ensure that the antisense strand is preferentially loaded into RISC).
The above correlations suggest that some not insubstantial amount of sense strand is making it into the RISC complex.
Here is the distribution of delta-delta-G for siPOOLs and siGENOME siRNAs targeting the same 500 human kinases (see bottom of post for discussion of calculation). A positive delta-delta G means that the sense end is more thermodynamically stable than the antisense end, favouring the loading of the antisense strand into RISC.
This discrepancy in delta-delta G is also consistent with comparison of mRNA knockdown:
The siGENOME knockdown data comes from 774 genes analysed by qPCR in Simpson et al. (2008). The siPOOL knockdown data is from 223 genes where we have done qPCR validation.
Of note, the siGENOME pools were tested at 100 nM, whereas siPOOLs were tested at 1 nM.
(It should be mentioned that, although consistent with the observed differences in ddG, this is only an indirect comparison, and delta-delta G is not the only determinant of functional siRNAs.)
Notes on intraclass correlation
Intraclass correlation measures the agreement between multiple measurements (in this case, multiple siRNAs with the same target gene, or multiple siRNAs with the same seed sequence). One could also pair off all the repeated measures and calculate correlation using standard methods (parametrically using Pearson’s method, or non-parametrically using Spearman’s method). The main problem with such an approach is that there is no natural way to determine which measure goes in the x or y column. Correlations are normally between different variables (e.g. height and weight). In a case of repeated measures, there is no natural order, so the intraclass correlation (ICC) is the more correct way to measure the similarity of within-group measurements. As ICC depends on a normal distribution, datasets must first be examined, and if necessary, transformed beforehand.
Robust methods have the advantage of permitting the use of untransformed data, which is especially useful when running scripts across hundreds of screening dataset features. The algorithm we use calculates a robust approximation of the ICC by combining resampling and non-parametric correlation.
Here is the algorithm, in a nutshell:
- Group observations (e.g. cell count) by the grouping variable (e.g. target gene or antisense seed)
- Randomly assign one value of each group to the x or y column (groups with one 1 observation are skipped)
- for example, if the grouping variable is target gene and siRNAs targeting PLK1 had the values 23, 30, 37, 45, the program would randomly choose 1 of the values for the x column and another for the y column
- Calcule Spearman’s rho (non-parametric measure of correlation)
- Repeat steps 1-3 a set number of times (e.g. 300) and store the calculated rho’s
- Calculate mean of the rho values from 4. This is the robust approximation of the ICC (rICC).
- Values from 4 are also used to calculate confidence intervals.
The program that calculates this is available upon request.
Notes on calculating delta-delta G
Delta-delta G was calculated using the Vienna RNA package, as detailed here: https://www.biostars.org/p/58979/ (in answer by Brad Chapman).
The delta-delta G was calculated using 3 terminal bps. We found that that ddG of the terminal 3 bps had the strongest correlation with observed knockdown. Others (e.g. Schwarz et al., 2003 and Khvorova et al., 2003) have also used the terminal 4 bps.
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