A Quantitative Cell-Based High-Content Screening Assay for the Epidermal Growth Factor Receptor-Specific Activation of Mitogen-Activated Protein Kinase

Ghosh, Rahul; Grove, Linnette; Lapets, Oleg

doi:10.1089/1540658042584215

Cited by 5 publications

(8 citation statements)

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“…The skewing of data, associated with whole population measurements, is a common issue between HCI, microarray analysis, and functional magnetic resonance imaging. 22 Logarithmic transformation is a common statistical technique used to stabilize population variation and eliminate skewed distributions found in data generated using technologies measuring large populations. 23 This transformation changes a multiplicative scale into an additive scale and reduces the influence of extreme values such as outliers caused by cell clumps.…”

Section: Resultsmentioning

confidence: 99%

“…20,21 A logarithmic transformation (base 2) was performed to avoid potential skewing of the data due to distribution errors and to stabilize the variance of the subpopulations. 22 Variability plots were then used to examine variation due to cell-to-cell, well-to-well, and plate-to-plate variation. Variance components were calculated to quantify the contribution of variation sources, and t-tests were then used to determine the statistical significance of observed distributional differences.…”

Section: Discussionmentioning

confidence: 99%

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High-Content Imaging Analysis of the Knockdown Effects of Validated siRNAs and Antisense Oligonucleotides

et al. 2007

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High-content imaging (HCI) provides researchers with a powerful tool for understanding cellular processes. Although phenotypic analysis generated through HCI is a potent technique to determine the overall cellular effects of a given treatment, it frequently produces complex data sets requiring extensive interpretation. The authors developed statistical analyses to decrease the time spent to determine the outcome of each HCI assay and to better understand complex phenotypic changes. To test these tools, the authors performed a comparison experiment between 2 types of oligonucleotide-mediated gene silencing (OMGS), antisense oligonucleotides (ASOs), and short, double-stranded RNAs (siRNAs). Although similar in chemical structure, these 2 methods differ in cellular mechanism of action and off-target effects. Using a library of 50 validated ASOs and siRNAs to the same targets, the authors characterized the differential effects of these 2 technologies using a HeLa cell G2-M cell cycle assay. Although knockdown of a variety of targets by ASOs or siRNAs affected the cell cycle profile, few of those targets were affected by both ASOs and siRNAs. Distribution analysis of population changes induced through target knockdown led to the identification of targets that, when inhibited, could affect the G2-M transition in the cell cycle in a statistically significant manner. The distinctly different mechanisms of action of these 2 forms of gene silencing may help define the use of these treatments in both clinical and research environments. ( Journal of Biomolecular Screening 2007:775-788)

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

High-Content Imaging Analysis of the Knockdown Effects of Validated siRNAs and Antisense Oligonucleotides

et al. 2007

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“…The number of ready-to-use assay kits provided by vendors is continuously growing. A rapidly increasing number of HCS assays that have been proven in screening projects, have been published, such as assays for analyzing mitotic spindle formation [5] , cell proliferation [6,7] , apoptosis [8] , migration [9,10] , nuclear translocation (e.g., GPCR-GFP [11] , NFAT-GFP [12] , or FOXO1-GFP [13] ), inflammation [14,15] , Wnt signaling for activation of β -catenin [16] , EGFR signaling [17] , function of gap junctions [18] , or neurite outgrowth [19] .…”

Section: Components For Hcsmentioning

confidence: 99%

High-content siRNA screening for target identification and validation

Krausz

Korn

2008

Expert Opinion on Drug Discovery

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Various methods have been established for the application of RNAi in mammalian cells also, which allows efficient and reproducible silencing of individual genes to gain functional information on each individual gene. Complex multi-parametric cell-based assays, combined with both technologies, provide an extraordinary valuable tool to help understand biological and pathological processes in a systematic way and discover new targets for pharmaceutical exploitation.

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“…Activation of the MAPK by the epidermal growth factor (EGF) receptor was quantitated using the nuclear localization of phosphorylated ERK by immunofl uorescence, together with internalization of the receptor ligand EGF, which was labeled with Texas Red. This assay allowed the identifi cation of compounds able to block activation of the MAPK pathway by a specifi c relevant oncology target [103].…”

Section: Kinasesmentioning

confidence: 99%

Drug Screening Using Cell Lines: Cell Supply, High‐Throughput and High‐Content Assays

Burger

Pöschke

Jurzak

2006

Drug Testing in Vitro

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A Quantitative Cell-Based High-Content Screening Assay for the Epidermal Growth Factor Receptor-Specific Activation of Mitogen-Activated Protein Kinase

Cited by 5 publications

References 0 publications

High-Content Imaging Analysis of the Knockdown Effects of Validated siRNAs and Antisense Oligonucleotides

High-Content Imaging Analysis of the Knockdown Effects of Validated siRNAs and Antisense Oligonucleotides

High-content siRNA screening for target identification and validation

Drug Screening Using Cell Lines: Cell Supply, High‐Throughput and High‐Content Assays

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