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Integrating HCS and RT-PCR, Researchers Enhance Gene Profiling and Validation for Drug Discovery

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High-content screening can be combined with real-time reverse-transcription PCR to increase the efficiency of gene profiling, characterization, and validation in drug discovery, according to research published by scientists from Johnson & Johnson.

Their findings could represent the first time that scientists have described how these two relatively young technologies can work together.

The research, published in the November issue of Methods, describes how J&J scientists developed a new screening strategy that combined high-content image-based screens on a Cellomics ArrayScan with Applied Biosystems' TaqMan quantitative RT-PCR method.

According to the authors, the method could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment.


"One clear advantage here is that you can operate with a significant degree of confidence. A fluorescent marker may tell you whether or not the pathway you're interested in is getting activated, and increase your confidence that your screen is working."

"With microarrays and bioinformatics, we have more and more markers that we can map along various pathways," Sergey Ilyin, bioinformatics/translational technology group leader at J&J and lead author on the paper, told CBA News last week. "One clear advantage here is that you can operate with a significant degree of confidence. A fluorescent marker may tell you whether or not the pathway you're interested in is getting activated, and increase your confidence that your screen is working."

Ilyin said researchers would also be able to combine HCS information from "one or several pathways you're interested in."

Since high-content screening is still in its infancy compared with other drug-discovery tools, the data it produces has only been meaningfully combined with a few other existing technologies. Most notably, HCS has found instant overlap with RNAi as a way to conduct high-throughput functional genomics screens and target ID and validation. In addition, researchers such as Robert Murphy at Carnegie Mellon University have begun to combine HCS with proteomics in an attempt to create image-based maps of protein location and activity inside of cells.

Combining HCS with gene-expression analysis, however, is still relatively unexplored. Ilyin said that this is the first paper his group has published on the combined method, and that he was unsure if any others had done so previously.

In the Methods paper, the J&J researchers used a Cellomics ArrayScan to monitor the trafficking of a GFP-tagged PKC-ßII protein in HEK cells. Using various algorithms, they were able to quantitatively assess fluorescence levels and specific protein localization. They followed these analyses with TaqMan RT-PCR studies on lysates from the same cells.

What's more, the researchers were able to determine the expression patterns of proteins from the cell lysates using a Ciphergen SELDI ProteinChip platform.

The method has a stand-alone value of being able to validate microarray findings, and also provides an opportunity "for practical applications of these microarray-derived markers in integrative screening strategies," the researchers wrote in the paper.

"We're able to look at several different dimensions, at the same time, in the same population [of cells]," Ilyin told CBA News. "One of these dimensions is high-content imaging, and in this case it was GFP-based, so one could engineer various fluorescent biosensors which could give you information either about activation of a specific transduction pathway, interactions with a specific receptor, the state of the cell, metabolism, cell cycle, et cetera.

"In the same cell population, you can look at another dimension, which is changes in the level of messenger RNA," Ilyin added. "From this dimension you can also reconstruct which pathways are being activated or being affected."

All in all, the work could help provide an integrated approach that could better illuminate signaling pathways in cells and further establish HCS as an important tool in the drug-discovery process.

"Applied along with functional genomics tools, this type of comprehensive assessment taking into account spatial and expressional changes may facilitate knowledge mapping of pathway systems and signal transduction network analysis, [particularly] in high-throughput experiments," the authors wrote.

One of the main caveats of the approach, Ilyin said, is that researchers must use live cells for the high-content screening step before lysing them for the RT-PCR step. Many HCS platforms are designed to work with live cells, but according to Ilyin, many people prefer to use fixed cells.

"The ArrayScan analysis has to be performed on live cells, and immediately after that, cells can be lysed," Ilyin said. "[After] you lyse the cells, you basically have quite a few options."

Shrinking the Experiment

J&J has been working intimately with both Cellomics and Applied Biosystems to develop the combined application, but Ilyin said that there is no official collaboration in place.

Furthermore, he said, the company has initiated a collaboration with Woburn, Mass.-based BioTrove in an attempt to optimize the RT-PCR step in the integrated approach. Specifically, the drug maker is working with BioTrove to reduce the volume of consumables — and thus the cost associated with such studies — and may be helping to adapt BioTrove's technology to live-cell applications.

BioTrove offers a platform called OpenArray, which is designed to use low sample volumes in a highly parallel fashion. Its potential applications include SNP analysis, RNA transcript analysis, and protein binding or enzymatic assays.

"We see significant value in RT-PCR assays, for the reason that you can take signatures from microarrays and apply them for compound screening, for efficacy," Ilyin said. "We were quite happy with the technology platform we had, which was [from] Applied Biosystems, and was sort of a gold standard.

"But a limitation here is that the cost is substantial," he added, "There are different ways to calculate that cost, but in any case, we're talking about several dollars per well. With that cost, it's difficult to go mainstream with RT-PCR, even though it offers a lot of advantages."

The BioTrove OpenArray platform is enabled by "Thru-Holes," which refers to a grid of 300-micron-deep holes on a standard microtiter plate. "It has over 3,000 wells, but most importantly, each well has a volume of about 30 nL," Ilyin said, which results in significant conservation of consumables. So far, he added, J&J has been very happy with initial results.

In addition, he said, his research group has developed a way to use the OpenArray platform in combination with HCS as described in the Methods paper, and even to adapt OpenArray to live-cell applications. BioTrove has not yet released a version of OpenArray for live-cell applications, though it has said in the past that it is a goal (see CBA News, 7/27/2004).

Ilyin said he could not provide further details about specific modifications the J&J scientists have made to adapt OpenArray to live-cell applications. He also said the research group is contemplating publishing data showing how the platform can be similarly used in combination with ArrayScan analysis.

— Ben Butkus ([email protected])

 

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