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.
Introduction Materials & Methods - Workflow
Using primary cells for RNAi based applications such as target identification or – validation, requires a highly efficient transfection displaying the essential steps of the automated Nucleofector® Process: technology in combination with a reliable and robust automation system. To accomplish these requirements we integrated the amaxa 1. Transfer of the cells to the Nucleocuvette™ plate, 96-well Shuttle® in a Tecan Freedom EVO® cell transfection workstation which is based on Tecan’s Freedom EVO® liquid handling 2. Addition of the siRNA, (Steps 1 and 2 could be exchanged), platform and include all the necessary components and features for unattended cell transfection. 3. Nucleofection® process, 4. Addition of medium, Count Cells 5. Transfer of transfected cells to cell culture plate for incubation ® Nucleofector Technology prior to analysis. 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. Nucleocuvette Plate Add Nucleofector +– The basic principle and benefits of the (empty) Solution Cell of interest Gene of interest Nucleofector® Technology 1 Nucleocuvette (cells) Transfer Cells Cells in Nucleofector > Enables highly-efficient transfection (18 µl/well) Solution Nucleofection® of difficult-to-transfect cell lines > Fast onset of gene expression 2 Nucleocuvette Transfer siRNA siRNA Plate (cells & siRNA) (2 µl/well) Cell type specific solution Gene transfer directly into the nucleus 3 Nucleofection 96-well Shuttle
Results 1 - Reproducibility 4 Nucleocuvette Add 80 µl Medium & Mix Medium (37° C) (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, 5 Nucleocuvette Transfer to Culture Cell Culture Plate demonstrating a high reproducibility. (cells & siRNA & medium) Plate
Nucleocuvette Plate
100 maxGFP™ expression - The analysis was performed on a (empty) 90 BD FACSCalibur™ 24h post Nucleofection®. The transfec- 80 70 tion efficiency of each well is shown per well of of a 96- 60 well Nucleocuvette™ Module. Column 4 contained two 50 Materials & Methods - Process Control 40 control samples receiving either no pulse or no plasmid. 30 The standard deviations for the single columns were 1.26 20 10 / 0.95 / 0.88 / 0.94% with an over-all standard deviation The entire process is controlled by Tecan’s Freedom 0 of 1.04% for all samples. EVOware® software, which allows Column Column Column Column 1 2 3 4 customized programming of your specific 1,6 1,4 SEAP expression - Cells were transfected with 1 µg protocols. All of the necessary steps for 1,2 plasmid encoding secreted alkaline phosphatase (SEAP). 1 transfection are automated, including: ® 0,8 24 h post Nucleofection SEAP activity was analyzed by a 0,6 colorimetric alkaline phosphatase assay. From the assay a 0,4 > Plating of cells to the desired density standard deviation of 0.08 (mean value = 1.24) was 0,2 > Overnight incubation and cell washing 0 calculated, resulting in an excellent coefficient of 12 3 4 5 6 > Re-suspension of cells and substrates prior variation (CV) of 6.9%. to Nucleofection® > 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.
100 100 90 90 Transfection Efficiency (left, maxGFP™ 80 80 expression) and Viability (right) in human T-cells 70 70 after pre-incubation of the cells in Nucleofector® 60 60 solution for up to four hours. The analysis was 50 50 performed on a BD FACSCalibur™ 24 h post 40 40 ® 30 30 Nucleofection . The mean efficiency and viability 20 20 from two Nucleocuvette™ modules is shown. 10 10 0 0 1234hours 1234hours
Results 3 - Highly Efficient Knockdown Summary & Conclusions
Efficient knockdown is demonstrated by data showing siRNA-mediated depletion of vimentin-mRNA in Jurkat and primary human Combining Tecan’s Freedom EVO® liquid handling workstation with amaxa’s Nucleofector® Technology allows for fully automated, T-cells. Cells were transfected with a siRNA duplex directed against endogenous vimentin. 24h post transfection vimentin mRNA reproducible and efficient transfection of difficult-to-transfect cell lines and primary cells, including T-cells. The system is ideal for levels were analyzed by RealTime PCR. Relative expressions compared to untreated control sample are shown. 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 Additional Benefits 80 70 Ease of use Optimized 96-well Nucleofector® Kits and protocols are available for many primary cells 60 and difficult-to-transfect cell lines. 50 Flexibility The technology allows for identical transfection conditions for various substrates including 40 Jurkat E6-1 DNA, siRNA and shRNA. 30 (ATCC®), TIB-152™ Relative expressions Safety The unique conductive polymer electrodes of the Nucleocuvette™ plates prevent metal ion 20 compared to untreated control sample release, and disposable plates minimize the risk of cross contamination. 10 (C, set to 100%) are shown. 0 1357911 13 15 17 19 21 23 25 27 29 C
100 90 amaxa AG, Europe/World amaxa Inc., USA 80 Scientific Support Scientific Support 70 +49 (0)221-99199-400 888-632-9110 (toll free) 60 [email protected] [email protected] 50 40 www.amaxa.com 30 Human T-cells 20 Nucleofector, nucleofection, Nucleocuvette, maxGFP and 96-well Shuttle are trademarks of amaxa AG. 15 samples compared to control (C, set to 100%) BD FACSCalibur™ is a trademark of Becton, Dickinson and Company. 10 ATCC® and the ATCC Catalog Marks are trademarks of ATCC used under License. Freedom EVO®, Freedom EVOware®, Tecan®, are shown. 0 and the Tecan logo are in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland. Not for use in 1 2344678910 11 12 13 14 15 C Cologne, June 2007 clinical diagnostics.