Transfection and Transformation - 2 Pages

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Transfection and Transformation

Catalog excerpts

ROBOTICS TECHNICAL NOTE Drug Discovery Automating the Process of Transfection-Reagent/ siRNA/Gelatin Solution Production for Genome Wide RNAi Screening Using Cellarrays The MitoCheck Project Group at the EMBL in Heidelberg which is one of the major sites of the Integrated Project MitoCheck “Regulation of Mitosis by Phosphorylation - A Combined Functional Genomics, Proteomics and Chemical Biology Approach” funded by the European Commission in the Sixth Framework Programme, carries out genome-wide loss of function screens using RNA interference and automated microscopy of human cultured cells to identify human genes required for mitosis. Authors Dr. Holger Erfle of Cell Biology/Cell Biophysics Programme and MitoCheck Project Group, EMBL Heidelberg/Germany, co-developed the method and coauthored this application note. Figure 2: The 96 channel head performs transfer of the transfection solution into the low volume 384 well plate. • Washstation for needles • Cooled source • Barcode reader • Standard 96 well plates • MICROLAB® STAR, 8 channels and 96-probe head • Low volume 384 well plate • iSWAP, internal plate handler • siRNAs • Transfection solution • Gelatin Protocol Method Figure 1: Fully automated system to perform the production of the transfection-reagent/siRNA/gelatin solution for genome wide RNAi screens based on the MICROLAB® STAR platform. The final pipetting is performed in a low volume 384 well plate. A major part of the reverse transfection cellarray method used in the MitoCheck Project RNAi screen relies on the reliable preparation of the siRNA transfection solution for the subsequent spotting of the array and – after drying – the seeding of the cells.

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To exclude artefacts from cross-contamination, negative controls were examined carefully. The phenotypic evaluation and basic fluorescence readouts did not show any carryover. Throughput and Capacity 384 reactions including incubation times were performed in 90 minutes. Figure 3: Automatic transfection on the STAR workstation using siRNA directed against polo like kinase (plk1) causing a penetrant mitotic arrest of the cell population. overview about how the preparation ot the transfection solution on the MICROLAB® STAR (Figure 1). • Resuspension of siRNAs in 96 well plates • Transfer of...

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