Researchers at the National Cancer Institute and Qiagen have used multiplexes of synthetic siRNAs corresponding to multiple gene targets to compress RNAi screen size in cell-based assays, according to a recent study.
They also demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as if each of the genes was targeted individually.
The findings are noteworthy because compressing an RNAi screen may cut assay costs, reduce reliance on liquid handling and automation, and accommodate limited-capacity equipment, said NCI researcher Natasha Caplen, the corresponding author on the paper, which appears online in the March 28 issue of Nucleic Acids Research.
“This technique can be used to look at multiple proteins within a complex, and to simultaneously study proteins in different pathways and networks,” said Caplen. “Having established that we could simultaneously silence multiple genes for gene function analysis, it occurred to us that we could also use this for potentially compressing the size of some RNAi screens.”
There are also biological advantages, according to Qiagen researcher and paper co-author Eric Lader. For example, there are instances where a gene-by-gene knockdown in a screen could give researchers a desired result, but there are other screens that clearly won’t work in this manner, Lader said.
He said these include screens where there are multiple related genes that overlap in function, or cases in which researchers want to screen knock downs of parallel pathways together, or knock down multiple genes in the same pathway to enhance the phenotype.
According to Caplen, “What we’ve done is lay out a methodology so that people can use it under very specific circumstances.”
This technique will only work in the context of an siRNA library with gene targets whose functionality is relatively diverse, she said. It is not going to work in assays with a relatively high hit rate, although it has a lot of potential in those with a low hit rate, Caplen added.
The study in the Nucleic Acids Research paper came about after Caplen used some of Qiagen’s siRNAs to demonstrate the feasibility of doing a multiplex screen.
The paper includes a “large amount of supplementary data that addresses some of the issues that we looked at when characterizing the gene-specific silencing mediated by synthetic siRNAs,” Caplen said.
“Multiplexing siRNAs corresponding to different genes can be used to look at multiple proteins within a complex, and to simultaneously study proteins in different pathways or networks. It also occurred to us that it could also be applicable for potentially compressing the size of some siRNA screens.”
She said other groups can use this data to look at how siRNAs against a broad range of cancer-associated genes are working, and what needs to be studied and characterized.
According to Lader, NCI and Qiagen have been working together in this area for four years, during which Qiagen has been designing siRNAs for the institute. The NCI has been trying to validate the functionality of these siRNAs and has been feeding the data back to Qiagen, he added.
Lader also mentioned that for RNAi, Qiagen has long-standing relationships with NCI, the Translational Genomics Research Institute, and researchers at the Dana Farber Cancer Institute.
For this technique to work properly, it is important to have as specific an assay as possible, according to Lader. If an assay is looking at cell division, many different factors could affect the final cell count in a well, such as genes that are involved in cell cycling, metabolism, or apoptosis.
If multiple genes at a time are being randomly knocked down, “there will be so many positives that it will be difficult to determine what is really important,” he said, although the Caplen group was able to identify some known and putative anti-cancer molecular targets in this way.
However, if the assay is looking at something as specific as blocking the translocation of a receptor protein from the cell surface to the nucleus when a hormone is added to the well, then if siRNAs are being multiplexed against multiple genes, its very unlikely that significant noise in the assay will be seen and complicate its interpretation.