I am new to this forum, so hello everybody! I am a postdoc in Amsterdam, the Netherlands. Background is Pharmacochemistry with some Molecular Biology.
The outer wells of 96 well plates used in the BioRad MyIQ Single-Color Real-Time PCR System give higher (=worse) Ct values than the wells in the middle of the plate. In some cases I have found a difference of more than 5 (!) Ct values between the corner wells and the inner wells.
I have tested this by performing RT-PCR, using the same sample in each well and the same primerset in each well, using BioRad Microseal B (MSB-1001).
Using BioRad semi-skirted plates (HSS9641) instead of generic non-skirted plates, or the BioRad Green Sealing Ring (170-8731) and/or BioRad Optical Pad (ADR-3296) seems to partially solve the problem, but there still is a difference of 1 Ct value between the inner and outer wells.
I have used another MyIQ machine as well, which showed a similar pattern.
Evaporation due to poor sealing was the first thing that came to mind, but after running several tests, it seemed that exactly the same wells are involved every time, ruling out a sealing problem.
Now I think that it has to do with the heating of the wells.
I have two questions:
1) Did somebody else experience these problems, and how were they solved?
2) What is an acceptable difference between Ct values of the same sample generated by a RT-PCR machine (leaving out pipetting errors)
Thank you for your answers!