I am working with DNA extracted from feces. I made a qPCR using concentrations of 100, 75, 50 and 20ng final. The CT value was very similar between dilutions between 29 and 30. My positive control has a CT of 23. I've done the primer efficiency and it's fine. Any reason for this to occur? From 100ng to 20ng should have some change. Since these are stool samples they may have inhibitors, but I don't know if they would affect this way
A gap of 1 CT between two samples represent a 2-fold difference in DNA concentration. Therefore, between 20ng and 100ng you shouldn't expect anything more than a difference of 2 CT. Have you made triplicates ? My guess is that such a "small" difference is often hidden within the standart deviation of qPCR replicates.
Also, did you get a CT value for your negative control(s) ?
A gap of 1 CT between two samples represent a 2-fold difference in DNA concentration. Therefore, between 20ng and 100ng you shouldn't expect anything more than a difference of 2 CT. Have you made triplicates ? My guess is that such a "small" difference is often hidden within the standart deviation of qPCR replicates.
Also, did you get a CT value for your negative control(s) ?
You say you've checked the primer efficiency. Did you do this via the serial dilution method? If so, is the assay still linear at around 29-30 CTs? Also, what is the CT of a negative control (e.g. a No-RT control)? I suspect you're outside the linear range of your assay.
What is ct value of negative control and what is status of melt curve.
Dear Marilia Cavalcanti , I agree with other. Unfortunately it is not clear for me which reason a really led to your troubles. But...100-20 ng it is only 5-fold dilution. I recommend you use 100 ng, 20 ng, 4 ng, 0.8 ng at least. When you have 3 cycles (3.32 in Eff = 100%) it is approximately 10 fold. I mean 100 ng - 10 ng step. In the case of 5 fold step you have to see about 2.3-2.5 (depending on reaction effectiveness) cycles between 100 and 20 ng.
Dear Dr Andrei, I am agree with your suggestion. It is better to do reaction with more dilution differences.
Dears,
Thanks for the answers!
- Martin Chenal , I made triplicates, the CT between dilution from 100ng to 20ng varied only 1 CT from 30.2 to 31.0. I made triplicates.
- Andrei S. Babenka, it really must have been a little difference, I can try to make more dilutions
- I did not use negative control, only the "blank" that instead of the sample contains water, the result was undetermined.
- Brian Bower , I did the primer efficiency and the result was 99%, the CTs went from 22.9 (200ng) to 34.5 (0.064ng), so having my sample results between 30 and 31
- The melting curve was good for the samples and similar to the positive control
If your assay was indeed linear to 34.5 CTs, that's rather good.
I'd still want to see a negative control reaction made using a full-dose of RNA but no RT as a control. Such a control will measure the amount of signal that is expected from any residual gDNA contamination. Since the gDNA contaminant is diluted when performing the linearity dilutions, the lowest point of your dilution series may not represent the true limit of detection for an undiluted sample.
I used to run a No-RT control (measures signal from gDNA), an No-Template Control (measures possible primer self-amplification) and a No-RT+No-Template Control to check for possible contamination of reagents. That might seem like overkill, but if you get a low CT for any of those controls you have a problem.
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