Abstract
BackgroundThe QuantiGene® Plex 2.0 platform (ThermoFisher Scientific) combines bDNA with the Luminex/xMAP magnetic bead capturing technology to assess differential gene expression in a compound exposure setting. This technology allows multiplexing in a single well of a 96 or 384 multi-well plate and can thus be used in high throughput drug discovery mode. Data interpretation follows a three-step normalization/transformation flow in which raw median fluorescent gene signals are transformed to fold change values with the use of proper housekeeping genes and negative controls. Clear instructions on how to assess the data quality and tools to perform this analysis in high throughput mode are, however, currently lacking.ResultsIn this paper we introduce QGprofiler, an open source R based shiny application. QGprofiler allows for proper QuantiGene® Plex 2.0 assay optimization, choice of housekeeping genes and data pre-processing up to fold change, including appropriate QC metrics. In addition, QGprofiler allows for an Akaike information criterion based dose response fold change model selection and has a built-in tool to detect the cytotoxic potential of compounds evaluated in a high throughput screening campaign.ConclusionQGprofiler is a user friendly, open source available R based shiny application, which is developed to support drug discovery campaigns. In this context, entire compound libraries/series can be tested in dose response against a gene signature of choice in search for new disease relevant chemical entities. QGprofiler is available at: https://qgprofiler.openanalytics.eu/app/QGprofiler
Highlights
The QuantiGene® Plex 2.0 platform (ThermoFisher Scientific) combines Branched chain DNA (bDNA) with the Luminex/xMAP magnetic bead capturing technology to assess differential gene expression in a compound exposure setting
To the best of our knowledge, clear instructions on how to assess the data quality are currently lacking, while most researchers perform the transformations in a local spreadsheet environment or by means of manual calculation. Against this background we present experimental data tested in dose response and introduce a newly developed open source available R based shiny application: QGprofiler, that allows for proper QuantiGene® Plex 2.0 assay optimization, choice of housekeeping genes (HKG) and data pre-processing from raw gene expression to normalized fold change (FC) values including appropriate QC metrics
Experimental conditions In order to assess the minimal number of beads, the stability of the HKG and optimize the data analysis flow, data from 304 samples were generated from five independent QuantiGene® runs (Table 1)
Summary
The QuantiGene® Plex 2.0 platform (ThermoFisher Scientific) combines bDNA with the Luminex/xMAP magnetic bead capturing technology to assess differential gene expression in a compound exposure setting. This technology allows multiplexing in a single well of a 96 or 384 multi-well plate and can be used in high throughput drug discovery mode. The QuantiGene® Plex 2.0 platform (ThermoFisher Scientific) combines bDNA with the Luminex/xMAP magnetic bead capturing technology This platform does not require an RNA extraction step, as it measures mRNA levels directly from cultured cells [10], cell lysates [11], tissue homogenates [12], formalin-fixed tissues [13], to name only a few starting points. HKG that fell outside the [0.8; 1.2] FC range were removed from the analysis, until the FC of the remaining HKG met this FC range criterion
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