Hi all,
I have treated the MDA cells with a drug, extracted the RNA by TRIZOL, then synthesized cDNA, which is done correctly, but the Real-Time curves do not look correct! (the following figures). I don't know why the curves aren't overlapping?
I repeat the test, but the results are the same.
Thanks for any help.
figure1. Blue peak is NTC, The rest of the peaks are for a primer in MDA cells treated with different doses
figure2. my positive control (B2M)
Well, the simple answer would be that...your PCR didn't work properly.
There are a host of reasons why this might be, but it's difficult to answer with the level of detail you've provided.
What were your Cq values, and how consistent were they?
How many replicates per sample? (qPCR should usually be done in triplicate)
How much cDNA did you use per replicate? (in volume or estimated ng)
Did you dilute your cDNA prior to use?
How much RNA did you use to make your cDNA?
What were your RNA 260/230 ratios like?
Looks like you may have a few things going wrong. First, there is a nice big peak in your no template control (NTC), that should not be happening. Either you are getting a primer dimer or there is some kind of gDNA carryover or contamination.
Also, as you mention, your peaks don't line up. Try running out your reactions on a DNA gel to see if you are getting the expected fragment sizes. Have you tried a dilution series with these primers to check their specificity and efficiency?
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