Rnai in Primary Cells and Difficult-To-Transfect Cell Lines

Automated High Throughput Nucleofection® RNAi in Primary Cells and Difficult-to-Transfect Cell Lines Claudia Merz, Bayer Schering Pharma AG, Berlin, Germany; Andreas Schroers, amaxa AG, Cologne, Germany; Eric Willimann, Tecan AG, Männedorf, Switzerland. <ul style="display: flex;"><li style="flex:1">Introduction </li><li style="flex:1">Materials & Methods - Workflow </li></ul>Using primary cells for RNAi based applications such as target identification or – validation, requires a highly efficient transfection technology in combination with a reliable and robust automation system. To accomplish these requirements we integrated the amaxa 96-well Shuttle® in a Tecan Freedom EVO® cell transfection workstation which is based on Tecan’s Freedom EVO® liquid handling platform and include all the necessary components and features for unattended cell transfection. displaying the essential steps of the automated Nucleofector® Process: 1. Transfer of the cells to the Nucleocuvette™ plate, 2. Addition of the siRNA, (Steps 1 and 2 could be exchanged), 3. Nucleofection® process, Count Cells 4. Addition of medium, 5. Transfer of transfected cells to cell culture plate for incubation prior to analysis. Nucleofector® Technology Remove Medium The 96-well Shuttle® combines high-throughput compatibility with the Nucleofector® Technology, which is a non-viral transfection method ideally suited for primary cells and hard-to-transfect cell lines based on a combination of buffers and electrical parameters. Add Nucleofector Solution Nucleocuvette Plate (empty) <ul style="display: flex;"><li style="flex:1">+</li><li style="flex:1">–</li></ul>The basic principle and benefits of the Nucleofector® Technology Cell of interest Nucleofection® Gene of interest 1Transfer Cells <ul style="display: flex;"><li style="flex:1">Nucleocuvette (cells) </li><li style="flex:1">Cells in Nucleofector </li></ul>Solution > Enables highly-efficient transfection of difficult-to-transfect cell lines > Fast onset of gene expression (18 µl/well) 2345Nucleocuvette (cells & siRNA) Transfer siRNA siRNA Plate (2 µl/well) Cell type specific solution Gene transfer directly into the nucleus Nucleofection 96-well Shuttle Add 80 µl Medium & Mix <ul style="display: flex;"><li style="flex:1">Nucleocuvette </li><li style="flex:1">Medium (37° C) </li></ul>Results 1 - Reproducibility (cells & siRNA & medium) Jurkat E.6-1 cells (ATCC® TIB-152™) were either transfected with pmaxGFP™ or a plasmid encoding secreted alkaline phosphatase (SEAP). The results shown reflect the excellent data quality and the absence of technical artifacts such as plate- or edge effects, demonstrating a high reproducibility. Transfer to Culture Plate <ul style="display: flex;"><li style="flex:1">Nucleocuvette </li><li style="flex:1">Cell Culture Plate </li></ul>(cells & siRNA & medium) Nucleocuvette Plate (empty) maxGFP™ expression - The analysis was performed on a BD FACSCalibur™ 24h post Nucleofection®. The transfec- 100 90 80 tion efficiency of each well is shown per well of of a 96- 70 60 well Nucleocuvette™ Module. Column 4 contained two 50 Materials & Methods - Process Control control samples receiving either no pulse or no plasmid. 40 30 The standard deviations for the single columns were 1.26 20 The entire process is controlled by Tecan’s Freedom / 0.95 / 0.88 / 0.94% with an over-all standard deviation 10 0<ul style="display: flex;"><li style="flex:1">EVOware® </li><li style="flex:1">software, </li><li style="flex:1">which </li><li style="flex:1">allows </li></ul>of 1.04% for all samples. Column 1 Column 2 Column 3 Column 4customized programming of your specific protocols. All of the necessary steps for transfection are automated, including: 1,6 1,4 1,2 1SEAP expression - Cells were transfected with 1 µg plasmid encoding secreted alkaline phosphatase (SEAP). 24 h post Nucleofection® SEAP activity was analyzed by a colorimetric alkaline phosphatase assay. From the assay a standard deviation of 0.08 (mean value = 1.24) was calculated, resulting in an excellent coefficient of variation (CV) of 6.9%. 0,8 0,6 0,4 0,2 0> Plating of cells to the desired density > Overnight incubation and cell washing > Re-suspension of cells and substrates prior to Nucleofection® <ul style="display: flex;"><li style="flex:1">1</li><li style="flex:1">2</li><li style="flex:1">3</li><li style="flex:1">4</li><li style="flex:1">5</li><li style="flex:1">6</li></ul>> Culture of transfected cells for further analysis. Results 2 - Robustness During the screening process the cells have to be stored in Nucleofector® solution for up to two hours prior to dispensing. The data shown, demonstrate the robustness of the main readout parameters efficiency and viability over time. 100 90 80 70 60 50 40 30 20 10 100 90 80 70 60 50 40 30 20 10 Transfection Efficiency (left, maxGFP™ expression) and Viability (right) in human T-cells after pre-incubation of the cells in Nucleofector® solution for up to four hours. The analysis was performed on a BD FACSCalibur™ 24 h post Nucleofection®. The mean efficiency and viability from two Nucleocuvette™ modules is shown. 00<ul style="display: flex;"><li style="flex:1">1</li><li style="flex:1">2</li><li style="flex:1">3</li><li style="flex:1">4</li><li style="flex:1">hours </li><li style="flex:1">1</li><li style="flex:1">2</li><li style="flex:1">3</li><li style="flex:1">4</li><li style="flex:1">hours </li></ul><ul style="display: flex;"><li style="flex:1">Results 3 - Highly Efficient Knockdown </li><li style="flex:1">Summary & Conclusions </li></ul>Efficient knockdown is demonstrated by data showing siRNA-mediated depletion of vimentin-mRNA in Jurkat and primary human T-cells. Cells were transfected with a siRNA duplex directed against endogenous vimentin. 24h post transfection vimentin mRNA levels were analyzed by RealTime PCR. Relative expressions compared to untreated control sample are shown. Combining Tecan’s Freedom EVO® liquid handling workstation with amaxa’s Nucleofector® Technology allows for fully automated, reproducible and efficient transfection of difficult-to-transfect cell lines and primary cells, including T-cells. The system is ideal for large-scale studies that involve high throughput transfection, such as RNAi-based screening for target identification and validation, or screening of cDNA libraries. % relative expression (% pulse only, sample 31 100 90 80 70 Additional Benefits Ease of use Flexibility Safety Optimized 96-well Nucleofector® Kits and protocols are available for many primary cells and difficult-to-transfect cell lines. 60 50 The technology allows for identical transfection conditions for various substrates including DNA, siRNA and shRNA. 40 Jurkat E6-1 30 (ATCC®), TIB-152™ Relative expressions The unique conductive polymer electrodes of the Nucleocuvette™ plates prevent metal ion release, and disposable plates minimize the risk of cross contamination. 20 compared to untreated control sample 10 (C, set to 100%) are shown. 0<ul style="display: flex;"><li style="flex:1">1</li><li style="flex:1">3</li><li style="flex:1">5</li><li style="flex:1">7</li><li style="flex:1">9</li><li style="flex:1">11 </li><li style="flex:1">13 </li><li style="flex:1">15 </li><li style="flex:1">17 </li><li style="flex:1">19 </li><li style="flex:1">21 </li><li style="flex:1">23 </li><li style="flex:1">25 </li><li style="flex:1">27 </li><li style="flex:1">29 </li><li style="flex:1">C</li></ul>100 90 80 70 60 50 40 30 20 10 amaxa AG, Europe/World Scientific Support amaxa Inc., USA Scientific Support +49 (0)221-99199-400 <a href="mailto:[email protected]" target="_blank">[email protected] </a> 888-632-9110 (toll free) <a href="mailto:[email protected]" target="_blank">[email protected] </a><a href="/goto?url=http://www.amaxa.com" target="_blank">www.amaxa.com </a>Human T-cells 15 samples compared to control (C, set to 100%) Nucleofector, nucleofection, Nucleocuvette, maxGFP and 96-well Shuttle are trademarks of amaxa AG. BD FACSCalibur™ is a trademark of Becton, Dickinson and Company. ATCC® and the ATCC Catalog Marks are trademarks of ATCC used under License. Freedom EVO®, Freedom EVOware®, Tecan®, and the Tecan logo are in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland. Not for use in clinical diagnostics. are shown. 0<ul style="display: flex;"><li style="flex:1">1</li><li style="flex:1">2</li><li style="flex:1">3</li><li style="flex:1">4</li><li style="flex:1">4</li><li style="flex:1">6</li><li style="flex:1">7</li><li style="flex:1">8</li><li style="flex:1">9</li><li style="flex:1">10 </li><li style="flex:1">11 </li><li style="flex:1">12 </li><li style="flex:1">13 </li><li style="flex:1">14 </li><li style="flex:1">15 </li><li style="flex:1">C</li></ul>Cologne, June 2007

Rnai in Primary Cells and Difficult-To-Transfect Cell Lines

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