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Month: September 2023

siPOOLs: robust reagents for gene silencing

siPOOLs: robust reagents for gene silencing

Although we talk a lot about off-targets, one of the main advantages of siPOOLs (complex siRNA pools) compared to single siRNAs or mini-pools (Dharmacon) is that they provide near optimum silencing of target genes. Two siPOOLs for the same gene give very similar knock down levels, and their silencing is around the best of any single siRNA. Given how many candidate siRNAs there are for a gene, and how difficult it is to accurately predict silencing levels, this makes siPOOLs the best choice for gene silencing.

The following plot, comparing independent siPOOLs and siRNAs for the same target gene, shows that siPOOLs for the same gene give more similar silencing than do siRNAs (these are Ambion Silencer Select siRNAs).

We see that the correlation for independent siPOOLs is nearly twice that for independent siRNAs.

(Note that for siRNAs we are doing all pairwise comparisons for 3 siRNAs per target gene. Randomly selecting 2 siRNAs per gene gives similar R values.)

In the above plot, we removed 3 siRNAs that did not work, for the gene TRIB1. TRIB1 has some association with the nucleus and has a short mRNA half life, both of which are factors associated with poor gene silencing.

The following plot shows the TRIB1 siPOOLs and siRNAs.

Note that including these non-functional siRNAs actually improves the reagent correlation, though not for a good reason!

We also see that independent TRIB1 siPOOLs give very similar silencing and it’s much better than for the siRNAs. In our experience, if a siPOOL does not work well for a gene, designing a second siPOOL does not substantially improve things, as the poor silencing is normally a feature of the target gene itself. ~50% silencing is probably about the best one can expect for this gene.

Just because siRNAs do not give any on-target silencing, this does not mean they can’t show up as hits in screening assays. Because most of the downregulation is in off-target genes (due to the seed effect), each of those TRIB1 siRNAs may silence nearly 100 genes.

We looked at a genome-wide RNAi screen that included these 3 Silencer Select siRNAs. We see that one of them gives a fairly strong phenotype (Z-score < -2 for cell count), even though the siRNAs do not silence their on-target gene.

Screening with siPOOLs is the smarter alternative, as you can be confident that they provide near optimal on-target silencing and have less off-target effects.

Cutting the Gordian Knot of RNAi off-targets

Cutting the Gordian Knot of RNAi off-targets

The C911 siRNA control generated a lot of excitement in the RNAi world when it emerged ~11 years ago. A former colleague, who was a pioneer in the commercialisation of RNAi, described it then as the biggest breakthrough in the last 10 years of RNAi research.

The idea of the C911 control is to get rid of the on-target effect of the siRNA by using the complement of bases 9-11, while retaining any off-target (seed-based) effects of the siRNA, which are mostly dictated by the bases in positions 2-8.

If the observed phenotype of the siRNA is due to an off-target effect (rather than silencing of the on-target gene), the C911 version will show the same phenotype. i.e., because it is not silencing the target gene, the phenotype must come from an off-target effect.

Despite the initial excitement, the C911 approach did not become that widely used. There are a number of drawbacks to the strategy, perhaps foremost being that new reagents must be ordered and the assay set up to run again. We’ve compared the validation of low-complexity RNAi reagents to the old lady who swallowed a fly.

The best strategy is to avoid getting entangled in off-targets in the first place. And that seems to be the approach preferred by the research community.

The following plot shows Google Scholar citations for siTOOLs (i.e., papers using our reagents) and the C911 method paper.

We see that after an initial adoption period, use of C911s tapered off and it has levelled out in recent years.

None of this suggests that C911s are bad. For single siRNAs or Dharmacon pools, they are indeed an effective control. But the inconvenience of the method has probably hindered its adoption.

The convenience and robustness of the siPOOL are its great advantages. The siPOOL approach ensures maximum on-target silencing and a minimum of off-target effects. We look forward to supporting more great research in the coming years.


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