Hello, I am trying to quantify some DNA viruses in human blood samples by comparing with standard curves made by serial dilutions of the same viruses. We have made nice standard curves with 100% efficiency, however all tested samples are giving same CT (25-26) which is not theoretically possible. NTC is always negative. please help to solve the issue.Thanks
Naveed
Is your melting curve clean (one peak)? How do you control for the absence of genomic DNA? If you are trying to amplify & quantify DNA from viruses, I don't understand your NTC. Can you explain?
May be you need to dilute more your samples to fit them in curve from serial dilution
may be presence of proviral DNA.
OR THE DETECTION LIMIT OF SYSTEM IS close to the dilution having max conc.
You don't have a very low Ct (corresponding to high sample concentration) so I hypothesize you have genomic DNA contamination. Have you used DNAse during RNA extraction?
Did you expect all the your samples to be positive ? This will likely depend on your sample your selection ? As Elizabeth pointed what is your negative control , the most appropriate would be a "dummy (water)" sample that has undergone similar processing as your samples from extraction to PCR. It is possible that what you seeing is contamination.
As others have pointed out there seems to be some type of contamination in your extraction reagents. Your NTC i.e. no template control is negative which indicates your amplification reagents are clean. Check all your extraction reagents. Do not open your calibrators in the extraction area.
All the best.
Do you use a probe or SYBR PCR? If SYBR - you probably need to read the fluorescence at higher temperatures (72 °C for example).
- Badges
- Science topic