I have a problem with dectection of human DNA using qPCR. I isolated DNA from human stem cells using DNA blood and Tissue kit from Quagen. Nanodrop measurements demonstrated 250 ng/ul concentration with the following purity parameters: OD ratio 260/280 = 2.08 and 260/230=1.92.
qPCR was done using quantifiler human DNA quantification kit (Applied biosystems). Only 7-8% of DNA could be recognized by qPCR. Do you have any ideas why does it happen? Possibly, undifferentiated cells are not well-detected by this kit? Or some technical problem? Standard curve was OK with slope -3.24 and R square 0.998.
How much DNA are you using as a template for the qPCR? more than ~10ng of DNA per reaction can inhibit the PCR. Also, make sure that the initial denaturation step is long enough and at the right temperature.
Hi Natalia,
Based on your 260/280 ratio (2.08), it sounds like you've got a high magnitude of RNA contamination in your DNA prep. Normally, a 260/280 ratio of ~1.85 is an indicator of pure DNA.
You can try to RNase your current samples and re-purify them. Keep in mind that the vast majority of the concentration you referenced is coming from the contaminating RNA.
David, I tried different template amounts starting from 115 pg and up to 100 ng. All the samples showed the same % of recovery.
Peter and Piming Zhao, thank you for the idea about RNA. During purification I used RNase supplied by the kit and thought it was OK. Possibly, I should run the sample on the gel. What should be % of agarose. Should I look for a ribosomal RNA? What size it should be?
Hi Natalia
I also think you should cross check your nanodrop DNA values with agarose gel results and see if they are matching...there can be problems associated with over estimation on nanodrop or also due to RNA contamination....you have used RNAse but didnot checked after the treatment...better check it on a gel
bye
Hi Natalia
I would like to know that what type of human stem cells you are using. Is it completely undifferentiated or in the process of differentiation? Since, stem cell differentiation is known to activate DNase in many cell types, could cause problems during qPCR. You should check the DNA integrity on Agarose gel, and make sure that your DNA is not fragmented.
Good luck!
If the purity rechecks do not turn up anything, I would suggest double checking your primers. In our lab we have found some primers with Tm values that are very specific. We had to add a second annealing step at a lower temperature to get the primers to anneal.
Thank you to all of you. After running DNA samples on the gel we found rRNA.
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