By Doug Macron
Despite the utility of high-throughput RNAi screening for gene discovery, the risk of false-positive and false-negative results remains an issue.
And while the former has been "a matter of extensive study," false negatives have received less attention, according to researchers from Harvard Medical School.
In a paper published last week in BMC Genomics, senior author and Norbert Perrimon and colleagues illustrated the problem by conducting a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens.
In conjunction with a focused RNAi screen, they determined that the rate of false negatives in such experiments is at least 8 percent and advise the use of "multiple, independently tested RNAi reagents per gene" to help address the issue.
According to Perrimon, RNAi screening is "a very powerful technology," but the rate of noise can obscure resulting data.
"This is an issue that everybody knows about, [but] very few actually address properly," he told Gene Silencing News. "The main reason is that it is very complicated and there is no" quick answer.
In terms of false positives, the problem stems from off-target effects, which cannot be predicted with perfect accuracy, he said.
In Drosophila screens, long dsRNAs are used to inhibit gene function, but the end product of their processing is unknown.
"We don't know if they make five or fifty siRNAs … and we don't know their respective concentrations," he noted. Meanwhile, in mammalian cell screens, the synthetic siRNAs used at high concentrations "can have thousands of targets because they work on the microRNA pathway.
"At the end of the day, the only way to know for sure is to do a rescue," Perrimon said. "The gold standard in the field [for both dsRNA and siRNA-based screens] is to … do some kind of genomic rescue of the effect.
"In practice, we don't do it because it's a lot of work, so [researchers have been] trying to come up with alternative strategies to remove more of those false positives in the experiments," he added. "That's why, in most cases, we try to use different reagents against the same gene."
On the other hand, "weak or ineffective" RNAi reagents can result in false negatives, according to the paper. Here, too, the problem may be mitigated by the use of multiple reagents per gene "as a single ineffective RNAi reagent would be compensated by those that are effective.
"An obvious caveat to this, however, is that simply by including more reagents, the number of false-positive results will also increase," the investigators cautioned in the paper.
Disambiguation strategies exist to deal with this problem, but in the paper Perrimon and his colleagues noted that, regardless of the approach used, "screeners must carefully interpret results obtained with multiple reagents per gene in order to reduce false-negative results without increasing the number of false-positive results to an unacceptably high level."
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