Friedrich-Alexander-Universität Erlangen-Nürnberg

High Throughput Biology & Screening Technologies

High Throughput Biology & Screening Technologies

High-throughput and High-content Screening technologies are successfully applied in academia, diagnostic medicine and the pharmaceutical industry to decipher intracellular signaling pathways, to discover diagnostic markers and to identify, characterize and prioritize molecules and genes relevant to homeostasis and disease. We develop technologies for high-throughput screening in cultured cells and computational methods to analyze phenotypic data sets, including the development of miniaturized assay formats and new approaches for massively parallelized microscopy.

Example: Multiplexing assay for decreasing false-positive rates in cell fitness screening

In drug and RNAi screening, one of the most commonly applied assay system is measuring the viability or fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. Multiplexing of different assaying methods and subsequent comparative analysis of multi-parametric data can accelerate hit confirmation compared to the classical approaches and may eliminate the need for additional re-testing using independent assays (Fig. 1). Further novel analytical solutions and technologies for high-throughput screening in life science are to be established through the MBT group within SAOT.

Figure 1. Work flow for cell fitness multiplexing assay in RNAi Screening. Prior to RNAi screening siRNA pools are plated into 384-well multititer plates (1) and stored at -20C until the experiment. For RNAi screening cells are seeded into the wells of multititer plates at defined density (2) and are reversely transfected for 72h. Upon reverse transfection of cells DNA stain Hoechst 33342 and the calcium indicator Calcein-AM are added to the cells in a total volume of 50ul and incubated for 1h at 37C (3). To reduce background fluorescence the staining solution is completely removed from the wells (4) and replaced by 10ul cell culture media without phenol-red (5). Calcein and Hoechst signals are measured using a plate reader (6). For assessing cell fitness via intracellular ATP level CellTiter-Glo CellTiter-Glo (CTG) is added to the cells and incubated at room temperature for 10 min (7). Luminescence intensity is recorded using a plate luminometer. Subsequent hit selection is conducted by combinatorial analysis of intersecting phenotypes. Modified from: Gilbert et al., PLoS One 2011 (accepted).

References:

Gilbert DF, Erdmann G, Zhang X, Fritsche A, Demir K, Jaedicke A, Muehlenberg K, Wanker EE, Boutros M (2011). A novel multiplex cell viability assay for high-throughput RNAi screening. PLoS One 2011, accepted.

Gilbert DF, Jaedicke A, Boutros M, Hochdurchsatz-Screening von Wirkstoff- und Signalweg-Interaktionen, Laborwelt, Nr. 1 / 2010 – 11. Jahrgang

Gilbert DF, Meinhof T, Pepperkok R, Runz H. DetecTiff: a novel image analysis routine for high-content screening microscopy. J Biomol Screen. 2009 Sep;14(8):944-55. Epub 2009 Jul 29. PubMed PMID: 19641223.

Bartz F, Kern L, Erz D, Zhu M, Gilbert D, Meinhof T, Wirkner U, Erfle H, Muckenthaler M, Pepperkok R, Runz H. Identification of cholesterol-regulating genes by targeted RNAi screening. Cell Metab. 2009 Jul;10(1):63-75. PubMed PMID: 19583955

Gilbert DF, Islam R, Lynagh T, Lynch JW, Webb TI. High Throughput Techniques for Discovering New Glycine Receptor Modulators and their Binding Sites. Front Mol Neurosci. 2009; 2:17. Epub 2009 Oct 30. PubMed PMID: 19949449; PubMed Central PMCID: PMC2782790

Mission

SAOT provides an interdisciplinary research and education program of excellence within a broad international network of distinguished experts to promote innovation and leadership in the areas

Optical Metrology
Optical Material Processing
Optics in Medicine
Optics in Communication and Information Technology
Optical Materials and Systems
and Computational Optics.