I am doing some qPCR and noticed that ct values for genes TNF, DPP10 and KCND2 are in the range 30s, I've tried a different cDNA kit which allows me use a total RNA of 5mcg and that has helped get the Ct values of TNF and KCND2 to 28~ My problem is I intend to do some RNAi assays and if my control is ~28 then my silenced genes will go above the 30s range and I might not be able to make sense of the results. DPP10 is still undetected. I've tried different primers for DPP10 (some in published papers) and still can't get any amplification. This is my first time using both DPP10 and KCND2 so I expected some challenges. The TNF however is what is confusing me. Shouldn't this be generously expressed by the cells? My cells are A549 and Beas2B cells. I need some help. Anyone know what I should do?
Hi Abiola
ct values are linked to the level of expression of your targets in your samples.
getting less RNA will give you a lower ct but you'll need to take into account for differential expression.
fred
Actually, Fred has it backwards. The few the copy numbers the MORE cycles it will take to see amplification.
If you want to see earlier amplification (lower ct values), then you need to start with more RNA.
Please use multiple primer sets for same gene. In my experience I found that different set of primers give different ct value for same gene. It depends on annealing parameters
Hi Katie A Burnette
oh yes of course you're right, giving more templates will mathematically give lower ct during RT-PCR.
fred
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