Topic pretty complex that will be treated in several posts.
Inhibition was already
discussed previously in « world of fluorophores part 2 » post about
most used molecules and the way to give a sample result considering inhibition
control results.
Nevertheless, here I will talk
about how to characterize inhibition from PCR results but also to get rid
of inhibition more or less and how to give a result considering inhibition
results.
Before any deeper analysis of this topic, a good thing is to
know whether your PCR runs have been even slightly subject to inhibition. Few hints
can help you :
- -
Imperfect sigmoid
amplification signal with :
o
Curve
flattening
o
Low raw
flurorescence level
o
Abnormal shape
of amplification curve
o
Exponential
phase of the curve widened or flattened
-
- Dilution non recovered by PCR results (eg. If you have less than 3.32 Ct between pure and 1/10 diluted sample, that probably means your sample is inhibited).
- Dilution non recovered by PCR results (eg. If you have less than 3.32 Ct between pure and 1/10 diluted sample, that probably means your sample is inhibited).
If you have some of these troubles in your PCR results, you may face
inhibition problems.
In next posts, I will try to give you personal experience about inhibition
in water and environnemental samples, pretty full of examples and sometimes
with solution found.
To me, I follow as much as I can standards about PCR development (French NF
T90-471 and ISO 12869). And to be clear about inhibition deal in PCR, I am not
keen of corrective factor to give a PCR result adding this factor to the sample
result. I prefer diluting samples to get rid of inhibition and give a result by
diluting corrective calculation.
Have good amplification and see you soon for inhibition and PCR.
