2 replies to this topic

[almost a doctor]

Hello, I'm having some issues with real time PCR and I was wondering if anyone here can help me. I'll try explaining my problem the best I can.
I work in an engineering company, and we are currently developing qPCR, of which unfortunately I can’t tell much for IP reasons. In brief just to put things in place, we don’t 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 can’t 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 doesn’t really explain why both the exponential and plateau part of the qPCR have shifted 10 cycles  !!        So, although I don’t think the problem is in the biology of the reaction I do have to agree with the engineer that there’s 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.

[gfischer]

almost a doctor, on Apr 22 2009, 07:59 AM, said:

Hello, I'm having some issues with real time PCR and I was wondering if anyone here can help me. I'll try explaining my problem the best I can.
I work in an engineering company, and we are currently developing qPCR, of which unfortunately I can’t tell much for IP reasons. In brief just to put things in place, we don’t 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 can’t 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 doesn’t really explain why both the exponential and plateau part of the qPCR have shifted 10 cycles  !!        So, although I don’t think the problem is in the biology of the reaction I do have to agree with the engineer that there’s 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.

If the sample hasn't changed on the ABI, and the band intensity on a gel is the same, I have to believe that the problem is with your in-house instrument.  Thee engineer may not want to hear it, but there's really no other explanation for what you're describing (except perhaps that the laws of biochemistry are different inside the new machine, you could try moving it to make sure it's not in some sort of space-time fissure ).

[aimikins]

there can be background fluorescence in the wells inside the machine. if you run the machine's self-tests you can get a reading and see which, if any, wells are affected.