I am trying to find RCN in real-time PCR on SYBR Green, but Cq among 3 repetition of the same DNA is going up and down around 1.2 cycle, so the error is too large. I'm working on genomic DNA extracted from buccal swabs, and have no way to measure its concentration, but I centrifuged the DNA before testing, diluted the DNA 100 times to reduce the concentration, but Cq continues to jump. Is there any way to handle this?
The three Ct values "from the same DNA": were they measured in the same run (plate) or in different runs (plates)?
What are the Ct values? Are they very low or very high?
Is the amplification specific (gel & melt curves)?
You don't get a lot of gDNA from buccal swabs. Diluting it 100-fold will leave you with even less, and the lower the [target] in your reaction, the more variable it will be.
I use buccal swab gDNA in qPCR, but I use ~0.33ul of undiluted column eluate per 20ul reaction (I make up a 60ul reaction + 2ul of gDNA and aliquot this between three wells). I typically observe Cq values in the 25-28 range, which indicates I don't have a lot of gDNA. My replicates are usually in very good agreement, however (i.e. it's low but readily quantifiable).
So try using more.
Can Kiessling Jochen Wilhelm Thank you for the answer. The CT value is between 24 and 27 cycles in a typical dilution, and increases to 27-31 with a 100-fold dilution (30 µl reaction). The values of Ct vary within the same run. According to the melting curve, the amplification is specific; nothing unnecessary is amplified.
Thanks for the update. Under optimum conditions ("perfect" amplification efficiency), the Ct should shift by about 3.3 cycles per 10-fold dilution (=log2(10)), hence about 6.6 cycles per 100-fold dilution. The shift will be larger under suboptimal conditions). If your normal samples have Ct-calues of 24 or larger, then it should not happen that 100-fold dilutions of these samples have Ct-values of less than 30 (you mentioned 27). This is strange.
Apart from this I don't see anything obvious that would possibly explain your findings. Does this happen only for a specific primer/assay or do you see this also for other primers? If yes, it might be due to the instrument. If only a particular set of primers/assay doesn't work well you might just design and order (sligtly) different primers, what might solve your problem.
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