I have been performing electroporation of HEK 293 cells as well as PBLs, and for this I followed a protocol that was established for a former PhD in my institue.
we use the linearized pGEM4Z-A120 as a template to generate the ivtRNA using the mMESSAGE mMACHINE T7 from Ambion, and use 20-25ug of ivtRNA to perfom the electroporation, but usually the amount of ivtRNA that we obtain is enough only for one or if we are lucky for two electroporations which has been become an issue due to the expensive cost of the Ambion kit.
So I was wondering whether if someone can explain me the criteria to select the amount of ivtRNA to do electroporation in particular of HEK 293 cells and PBLs that are the cell lines that I´m working with at the moment, because some literature mention that for PBLs you can use 2ug /1x106 cells and some others use even more, it will be great if I can maximize the use of my ivtRNA in order to save the kit which is expensive.
I use a Bio-Rad GenePulser Xcell to do the electroporation as follow for each cell type:
900V 2.8ms in 4mm cuvette for PBLs
400V 8ms in 4mm cuvette for HEK cells
for both I use between 1-2 million cells (usually 1.5 million per experiment)
I will appreciate any observation and suggestion very much, and it will be great if someone who has the experience with this protocol could advice me about this situation, thank you very much in advance.
We examined electroporation to express rotavirus RNA. To test the transfection efficiency of the specific cell lines (MA104, HEK 293H, BSR, Vero) we used plasmid eGFP. We used 1 million cells and found that the smaller cuvettes (1mm) were more effective in introducing and expressing larger amounts of eGFP. Cells were electroporated in the presence of 1 μg eGFP plasmid using the GenePulser Xcell (Biorad) using the same conditions you described (I would recommend using between 0.5-4ug of RNA/million cells). The expression of eGFP ranged from 20 – 40% (cells expressing eGFP) and was substantially lower than what was observed for the chemical transfections (between 60 – 95% eGFP expression in most cell types). Also, in our hands, significant portions of the HEK cell population were damaged during electroporation. Electroporation proved to be ineffective in expressing plasmid derived eGFP and caused considerable cell death. We could also not show any expression from viral RNA introduced by electroporation.
We also tested a wide range of transfection reagents. XtremeGENE HP (Roche) proved to be the best suited to successfully transfect and express in vitro derived rotavirus transcripts over a broad range of cultured cells (HEK cells in particular show a very high transfection efficiency). The XtremeGENE HP transfection reagent is formulated to transfect “hard-to-transfect” cells and was sufficient to successfully transfect 0.5-4 μg of transcripts. If your experiment allows it, I would recommend using this chemical transfection method.
Good luck.
In my opinion, Lipofectamine 2000 is one of good choices for transfection of RNA into cell lines (e.g., HeLa, 293T, Huh-7, etc.) instead of electroporation.
Im my early time, I had tried to transfect some kinds of in vitro transcribed RNAs (e.g., luciferase-expressing mRNAs) into human cell lines by eletroporation. To do this, much amount of in vitro transcribed RNAs as well as great numbers of cells should be prepared for the transfection. Moreover, it appeared to be less efficient than I expected. So, I'd changed my approach. I had utilized some kind of transfection reagents. Among them, using Lipofectamine 2000 had led to good and reproducible results in most cell lines I have ever tested. For example, Lipofectamine 2000-mediated transfection of capped and poly(A)-tailed Luc mRNAs (20~100 ng RNA and 1~2 ul Lipofectamine 2000 per well in a 24-well plate) into HeLa cells for 19 h gave rise to over 100,000 RLU activity, when I did luciferase assay using 1 ul volume from 100 ul of total lysates dissolved by 1x passive lysis buffer. RNA transfection into 293T cells showed higher Luc activity than HeLa in my hands.
If electroporation is not your only solution, therefore, I recommend using another transfection reagents for RNA transfection.
In addition, in vitro synthesis of RNA following conventional protocols is inexpensive and more applicable if you always need high amount of RNAs for your own purposes. Here I show the address for conventional in vitro transcription protocol (particularly, in vitro transcription using T7 RNA polymerase) :
http://molbio.mgh.harvard.edu/szostakweb/protocols/transcription/
And I attached my protocol for you. I usually obtained 20~30 ug of RNAs.
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