I am utilizing a pcDNA3.1 vector and gibson assembly to insert my gene of interest into HEK293T cells using lipid based transfection. I am doing some optimization with a control vector that expresses EGFP to determine what amount of time/charge ratio/confluency produces the highest protein output. I traditionally used immunofluorescence to measure transfection efficiency but I am trying to do something more quantitative. We do not have a flow cytometer at the moment so I am going with qPCR. How would I begin to set up an assay to measure transfection efficiency of my EGFP plasmid? I am completely new to qPCR so I really need help from the very beginning. Thanks!
Using qPCR or immunoblotting are not a good method for determining actual transfection efficiency as these both provide aggregate expression levels for all of the cells in your experimental condition. Simply counting the number of GFP+ cells per field using fluorescence microscopy will provide much more informative data.
However, it sounds like you're more interested in maximizing transgene protein production so Western blotting is probably going to be the most useful approach for you. You could do qPCR, but this is not going to be as informative since protein levels are not consistently correlated with transcript levels.
If you want to perform qPCR for this, the first step will be collecting and purifying the RNA from your transfected cells. Generally this should be done at 24 - 48 hours after transfection. You MUST perform DNase treatment on your RNA since the CDS of EGFP in the plasmid will be indistinguishable from EGFP cDNA. I am a big fan of the Qiagen miRNEasy kits for RNA purification, but there are wide variety of kits (and DIY recipes) available.
After you've purified your RNA, you'll need to perform first-strand/cDNA synthesis. Again there are a large number of kits available for this process. The primary choice you'll need to make at this step is using random hexamers, oligo-dT, or gene specific primers. Generally I would recommend oligo-dT if you're interested in protein coding genes since they will have a poly-A tail.
Once you've made your cDNA you're finally ready to run the qPCR. Here you can use either a SYBR Green system (where you will have to design your own primers or find published ones) or TaqMan probes (commercially available primer pairs and fluorescent probes). In addition to measuring the expression of EGFP/your GOI, you will also need an endogenous control (such as GAPDH, beta actin, etc.) to allow you to compare expression between your samples. It will come as no surprise that there are also a large number of kits that can be used for this step.
This Youtube series from BioRad Laboratories provides a fairly comprehensive overview of qPCR experimental design and protocol. https://www.youtube.com/watch?v=YPKJbK2axQk&list=PLrAEgIY86I6zbxvXgXTfL-CtI0A7C8sD_&index=7
ThermoFisher also has a good amount of learning material on qPCR that you might like to take a look through: https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center.html
I agree with the detailed response from John Hardy Lockhart which covered all aspects of the question. Given the statements of the question description, since measurement of protein output is the goal for this optimization, I think western with GFP antibody (your control) and any standard (such as GADPH or actin) will inform you about the best conditions for your transfections. Best of luck.
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