My experiment consists of electroporation of PBMCs (peripheral blood mononuclear cells) with plasmid DNA and use of modified cells 4h after electroporation. However, after 4h I still won´t have protein expression, but I need a reporter to show that indeed the cells were electroporated and the plasmid entered the cells. Also, it would be good to have a reporter as the electroporation efficiency will probably vary between experiments, so I can control this.
I thought about using a small DNA (ss or ds) conjugated to Cy3 or Cy5, so I can check by flow cytometry. However I´m afraid that, being smaller compared to plasmids, this will transfect almost 100% of the cells and will not correlate with plasmid transfection efficiency. Any ideas or alternatives on this?
At 4 hours post transfection, it is unlikely that your cells would be generating any RNA from the plasmid you put into the cells. What is the aim of this, my I ask? After 12-18 hours you can start to see differences in RNA expression from the plasmid, but 4 hours is relatively too short.
As answered by Craig, you can not expect expression before 10 h post electroporation and it is even difficult with PBMC. Since your aim is just to know whether the plasmid entered cells or not, I would suggest to do a qPCR from the total DNA from the electroporated cells (you need to have a controls where DNA is added but not electroporated to know the surface binding of DNA. And also, do not forget to wash the cells to remove free DNA from the sample).
As above - just to add - as a control a GFP reporter (in the plasmid you are using) is the general method for checking transfection/electroporation levels. Indeed you can tag this to your protein (at C terminal but remove the stop codon) so it will be roughly same size (and the same function).
One other thing - I generally let PBMC "revive" overnight in media if they are from LN2 storage (i.e. add 100 million to 100 ml of RMPI 10% FBS and stand upright in T75 in incubator overnight). Although they won't proliferate this should ensure the PBMC have a decent amount of metabolic activity and placing upright minimises amount sticking to the plastic of the flask.
@Craig: I know this. I just need a way to compare between different experiments how much plasmid DNA enters the cell. If I don´t measure it and somehow in one experiment I had a problem during electroporation, I will have a huge variability.
@Ramachandramouli: I had the same idea, but it is not practical. I need to use the cells 4h after electroporation, I can´t wait for the PCR results.
@Gary: I think you didn´t read the question...
Also, per Ramachandramouli suggestion. Set up the experiment in duplicate. @4h use one set for you experiment. Use the second set for the qPCR in parallel. So if you have discrepancy in your experiment, you can compare it with your pPCR results to see if it is real or false.
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