I am trying to see if DNA purification step will increase nematode detection in soil DNA. Soil DNA was extracted and half of the volume was used as it is (raw DNA extract) whereas remaining half was purified using commercial soil DNA purification kit. I ran qPCR using SYBR Green qPCR master mix with both raw DNA and purified DNA. Cq values for raw DNA extracts were higher compared to purified DNA as expected. However, when same DNA samples were run in ddPCR with Universal SYBR Green Supermix ( specific to ddPCR) , the copy numbers detected in raw DNA extracts were higher than the purified DNA samples. Since purified DNA samples had lower Cq values in qPCR, I was expecting higher DNA copy numbers in ddPCR but the results were different. I understand SYBR green is not specific to target DNA, but still, is it possible a sample to have a higher Cq value in qPCR and higher DNA copy numbers in ddPCR compared to a sample with a lower Cq value in qPCR and at the same time lower copy numbers in ddPCR?
Hi Bisho. Inhibition may be in play here. It is known that ddPCR is significantly less prone to inhibition compared to qPCR. So, probable scenario is: the raw samples have higher DNA amount (also more inhibitors) and perform poor in qPCR (inhibition) and better (no inhibition) in ddPCR compared with purified samples (less DNA amount and less inhibition- OK for qPCR,). In order to prove that you try dilutions of raw samples in qPCR. Also measuring the DNA quantity (preferably dsDNA with Qubit) in both raw and purified samples would give you a hint. Hope this helps.
Thank you, Dr. Mavridis! I will run qPCR and ddPCR with diluted samples and will see if the results vary.
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