I work in an engineering company, and we are currently developing qPCR, of which unfortunately I canít tell much for IP reasons. In brief just to put things in place, we donít use a commercial instrument (although we do have an ABI 7000). Both the thermocycling and the optical side of our system have been built in house. Until recently we were getting Cts of ~18-22 for a particular sample (very similar to what we get in the ABI instrument). However this has now changed and we canít see anything happening before cycle 28 (our protocol runs 30 cycles). If we run a 40 cycles PCR, the curve looks exactly the same as it used to but with a 10 cycles shift (Cts of ~28-32).
Now, these same samples still behave the same on the ABI instrument, and when analysed by electrophoresis and I'm stuck for ideas.
The engineer blames the reaction which of course I (the scientist) believe to be fine. I on the other hand blame the optics, and have managed to show that they are less sensitive than they used to. However, this doesnít really explain why both the exponential and plateau part of the qPCR have shifted 10 cycles !! So, although I donít think the problem is in the biology of the reaction I do have to agree with the engineer that thereís something odd going on that cant just be explained by the optics being less sensitive than they were.
Hope this makes sense and someone out there has any ideas for me.
Your help will be much appreciated.
Edited by almost a doctor, 22 April 2009 - 04:59 AM.