RNAi Microarray Analysis in Cultured Mammalian Cells

Abstract

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent cells, incubated to allow reverse transfection, and assessed for the effects of gene silencing by digital image analysis at a single cell level. Validation experiments with HeLa cells stably expressing GFP showed spatially confined, sequence-specific, time-and dose-dependent inhibition of green fluorescence for those cells growing directly on microspots containing siRNA targeting the GFP sequence. Microarray-based siRNA transfections analyzed with a custom-made quantitative image analysis system produced results that were identical to those from traditional well-based transfection, quantified by flow cytometry. Finally, to integrate experimental details, image analysis, data display, and data archiving, we developed a prototype information management system for high-throughput cell-based analyses. In summary, this RNAi microarray platform, together with ongoing efforts to develop large-scale human siRNA libraries, should facilitate genomic-scale cell-based analyses of gene function.

Cell microarrays and RNA interference chip away at gene function

“…Mousses et al developed a similar method with the goal of highthroughput RNAi experiments rather than siRNA probe selection 13 . They arrayed rhodamine-labeled siRNA targeting eGFP in a lipid transfection reagent onto glass slides and demonstrated potent and localized knock-down of eGFP expression in a lawn of HeLa cells stably expressing eGFP.…”

“…Given the apparatus necessary for screens in 96-or 384-well plates, high-throughput loss-of-function screening would be simplified by the use of cell microarrays for large RNAi experiments [7][8][9][10][11][12][13][14][15] . RNAi cell microarray slides should hold at least 5,000-6,000 spots of RNAi reagents, enabling whole-genome screens on a small number of slides 8,15 .…”

“…Efforts by a number of groups to develop cell microarrays using RNAi have introduced the possibility of using cell microarrays for largescale RNAi studies [7][8][9][10][11][12][13][14][15] . Although this technique is still in its infancy, cell microarrays can considerably reduce the amount of effort necessary for rapid cell-based RNAi screens.…”

Substrate-mediated delivery from self-assembled monolayers: Effect of surface ionization, hydrophilicity, and patterning

“…Plated cells were transfected and could be analyzed for cellular responses using a variety of imaging or biochemical techniques. These systems have also been used for transfection of siRNAs, which were printed in cationic lipid/Matrigel mixtures [49]. Alternatively, Chang et al [50] developed a technology they refer to as "surfection", in which a cationic polymer (PEI) and collagen are coated onto a surface.…”

“…Also, DNA complexes have been mixed with fibronectin and spotted onto glass slides for arrayed transfection of human mesenchymal stem cells [51]. While transfected cell arrays hold great potential [46][47][48][49][50][51], further development of a substratemediated delivery system that efficiently transfects a wide variety of primary cells and cell lines, while allowing for spatially-controlled delivery within the different domains is required [46,52,53]. Using SAMs on gold to form transfected cell arrays can allow precise control over surface chemistries, interactions between the substrate and DNA complexes, transfection efficiencies, and pattern sizes to create a well-characterized and efficient array delivery system.…”