I was wondering if someone has detected the mRNA levels of the FGF21 in Hepg2 cells at serum-starved state with low or high glucose by qPCR ?
I have used a new primer set, that bioinformatically speaking, looked promising. First I did a gradient qPCR to find the optimal annealing temperature, which was found to be 54C. Then the qPCR was done in 3 steps, with annealing at 54C for 35s and extension at 72 for 30s on ABI's StepOnePlus.
However, what I get by qPCR is what is attached - the Cq for most amplification curves is well beyond 30 (with cDNA input of about 2ng in RNA equivalents per reaction). The good news is there is only one peak in the melt curve and only one band in the 4% agarose. The bad news is I don't have access to human liver cDNA, where this mRNA is known to be very abundant in order to cross-check the primers, so I am hoping someone tried to detect it in Hepg2?
If you are VER VERY careful, you can generate a standard from a conventional PCR. If this is your aim.
Thank you for your reply Jochen. You're right - I could extract the aplicon from the gel band, purify and quantify the DNA to estimate the copy numbers and then to prepare a standard curve. I did this with other amplicons and it works great. However I was actually expecting a quite high expression of this gene in Hepg2, as another primer set for FGF21 for mouse in liver gave me Cqs around 22-23. So before estimating if the qPCR assay for the human FGF21 is linear behind Cq 30 (in which case it is ok to use the Cqs I already have), I was just wondering if this low expression in "normal" in HepG2.
Under normal physiological condition FGF21 mRNA level is not overexpressed in HepG2. I have tested it by realtime-PCR several times.
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