I've done several techniques to transfect shRNA-containing plasmid into A549. The techniques that i've done including calcium phosphate, lipo 3000, neon transfection. Now i planned to try electroporation but couldnt find any literature that already done electroporation on A549. Have anyone try this before? I need the parameters; voltage, no. of pulses, pulse duration, cell density
If you're having problems with transfection efficiency I would recommend finding a transfection reagent specific to A549 (https://altogen.com/product/a549-transfection-reagent-lung-carcinoma-ccl-185/). Electroporation is generally only more efficient in suspension cell lines like Jurkat, but for lung cancer lipofection is generally going to give better results, especially with pre-optimized protocols.
If you are going to use nucleofection (electroporation), see the details below in the link.
Link: http://bio.lonza.com/fileadmin/groups/marketing/Downloads/Protocols/Generated/Optimized_Protocol_340.pdf
I have not done electroporation with A549 but have with other cells. I believe our lab has used this technique with A549 though. I did this using siRNA but shRNA delivered by electroporation has been seen in other publications.
Cells were "suspended in the DMEM medium at 1X10^7 cells/mL, and mixed with 200 nM siRNA. Cell/siRNA suspensions in 0.1 cm cuvettes were then electroplated using ECM 830 square wave electroporation system (BTX) at 920 V, 120 mF, 2 pulses at 0.1 s interval. After 15 min at room temperature, cells were transferred to 100 mm dishes (Corning) containing DMEM complete media, and incubated for 72 h."
The cell concentration when electroporated is very important but you don't have to have a lot for each treatment.
Mix your cells with siRNA in eppy tubes to make sure it is well mixed and then transfer to cuvette. After electroporation leave cells in cuvette for the 15 minutes. Gently transfer the cells to culture plate and use some of the media to again gently wash the cuvette out and add back to plate. Our cuvettes came with tiny wide bore bulb pipettes which are more gentle than a regular pipette tip.
I have done with SH-SY5Y.
Scott, T. L., Wicker, C. A., Suganya, R., Dhar, B., Pittman, T., Horbinski, C. and Izumi, T. (2016), Polyubiquitination of apurinic/apyrimidinic endonuclease 1 by Parkin. Mol. Carcinog.. doi:10.1002/mc.22495
Even if or when you find conditions for electroporation of your specific cells, use the method only as a rough estimates. You will have to do your own optimization with your setup for optimum results.
If you're having problems with transfection efficiency I would recommend finding a transfection reagent specific to A549 (https://altogen.com/product/a549-transfection-reagent-lung-carcinoma-ccl-185/). Electroporation is generally only more efficient in suspension cell lines like Jurkat, but for lung cancer lipofection is generally going to give better results, especially with pre-optimized protocols.
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