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EMBL Team Develops New RNAi Screening Platform Using Video Imaging; Tech Available to Researchers


A team of researchers from the European Molecular Biology Laboratory published this week in Nature Methods details about a newly developed automated platform for high-throughput RNAi screening that incorporates video imaging in order to overcome the limitations of existing high-content RNAi screens.

The Heidelberg, Germany-based institution has also begun collaborating with researchers from other European nations to make the technology widely available, according to Jan Ellenberg, an EMBL researcher and co-author of the Nature Methods paper.

"After the completion of the human genome sequencing project, the task of functional genomics is to discover protein function genome-wide," the researchers wrote in the paper. "Currently, RNAi is the method of choice to study loss-of-function phenotypes in human cells by specifically suppressing the expression of virtually any desired protein-coding gene."

While several RNAi screens of human cells have been reported, these have "typically been based on endpoint assays of cells transfected in microtiter plates," the researchers added. "This allowed reasonable throughput but limited the information content of the phenotypic readout."

"Being microscopists, we said, 'In cell biology, what you do if you look at things that change over time is you just take a video of the cells over the duration that it takes to carry out the process and you use that for screening.'"

In doing a large-scale genome-wide experiment, "most people just chose a certain time point after RNAi knockdown to look at the phenotypes they are interested in … because of experimental limitations," Ellenberg told RNAi News this week. However, "phenotypes arise at different points in time and they are usually not static."

As a result, if an assay is run shortly after gene knockdown, it is usually very specific, but "you only get the phenotypes of proteins that are relative labile and are run down quickly — you only get part of the answer you are looking for," he said.

Conversely, assays run at late time points are more comprehensive but lead to specificity problems since it becomes difficult to differentiate between primary consequences of knockdown and secondary effects for rapidly turned-over proteins, the paper's authors wrote.

To tackle the problem, Ellenberg and his colleagues turned to video imaging to develop a "time-resolved" way of scoring phenotypes in large-scale RNAi screens.

"Genome-wide screening means you do on the order of 20,000 separate experiments, and phenotypic scoring is a lot of work even if you do it in microtiter formats or [on] microarrays," he said. "If you want to do this in a time-resolved fashion over several days you have many more data points you need to collect … [and] you deal with issues like cell synchronization and reproducibility at different time points.

"Being microscopists, we said, 'In cell biology, what you do if you look at things that change over time is you just take a video of the cells over the duration that it takes to carry out the process and you use that for screening,'" Ellenberg said.

"Limitations in manual data acquisition of live movies as well as manual, and thus inherently biased and nonquantitative, data annotation have so far precluded the use of live-cell imaging for RNAi screening in vertebrate cell systems," the researchers stated in the Nature Methods paper.

However, recent technological advances have cleared a path around these hurdles. The researchers took "advantage of the miniaturized RNAi delivery offered by transfected cell microarrays in which individually spotted siRNA transfection mixes are directly taken up from the solid phase by cells seeded on top of the array."

By spotting siRNA microarrays in live-cell imaging chambers, "we were able to perform time-lapse microscopy of HeLa cells on the arrays," the researchers wrote. "By massively increasing the throughput of fluorescence imaging and developing computerized analysis of the phenotypes by digital image processing, we established a fully automated high-throughput and high-content workflow of RNAi screening by time-lapse imaging."

"The innovation really is the combination of video microscopy and genome-wide RNAi screening," Ellenberg said. "Those two things have typically been very opposite types of technology. Video microscopy has been a one-gene interrogation where you look in a lot of detail and spend many months on a single gene to characterize it by detailed imaging and high-content microscopy. On the other side has been [the] genome-wide screen where you use very superficial scoring methods to do things rapidly because you have to be fast if you look across the entire genome," he noted.

"Being microscopists, we said, 'In cell biology, what you do if you look at things that change over time is you just take a video of the cells over the duration that it takes to carry out the process and you use that for screening.'"

"It's really combining these two things — one is more a cell biology technique and one is more a genomics technique," he added.

Ellenberg pointed out that the success of the screening system — which was validated in a pilot screen assaying cell division — is based not only on advances in microscopy but advances in "dealing with the kind of data you get from video imaging if you do it on such a large scale. It's a huge amount of pictures that you need to analyze, and [the Nature Methods paper described the] method of how the computerized imaging process is absolutely essential to extract the phenotypic data from the videos."

Although the RNAi screening method involves technologies not readily available to the average researcher, Ellenberg anticipates it is within reach of the major life science research institutions, which often open their facilities to visiting scientists.

For example, "at EMBL … visitors come to use [the] central infrastructure," he said. "EMBL each year has 2,000 scientists visiting, not only for screening but all kinds of things. This screening technology will also be available for other European scientists at EMBL, and we're already collaborating with people from different countries who want to take advantage of the technology."

— Doug Macron ([email protected])

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