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differently behavioring replicates in real-time PCR


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#1 egerecske

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Posted 08 September 2009 - 01:27 AM

Hi all

I have a problem with my HRM studies. Many of my replicates are very different from each other - I mean the replicates themselves are different. I figured out tha there is also a big difference in fluorescence level at the start of HRM. Cheking the Cp values, the difference between replicates are ranging from 0,06 to 12,63. a robot is pipetting the reactions, but I still cannot uinderstand, how can be such a big difference between replicates both in Cp and end-fluorescence. Even in replicates, which one has a Cp around 20-25, and itīs paralell has 30-35, many times the lower Cp has the lower end-fluorescence, which is right the opposite of what Iīd think. Can anyone give me an explanation for this, and what shall I try to solve the problem...?
In some plates everything works well, and in others not, so itīs not a permanent problem, but I test 400 samples in a run so itīd be very important for me to find a solution.
thanks

#2 almost a doctor

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Posted 08 September 2009 - 02:44 AM

Hi all

I have a problem with my HRM studies. Many of my replicates are very different from each other - I mean the replicates themselves are different. I figured out tha there is also a big difference in fluorescence level at the start of HRM. Cheking the Cp values, the difference between replicates are ranging from 0,06 to 12,63. a robot is pipetting the reactions, but I still cannot uinderstand, how can be such a big difference between replicates both in Cp and end-fluorescence. Even in replicates, which one has a Cp around 20-25, and itīs paralell has 30-35, many times the lower Cp has the lower end-fluorescence, which is right the opposite of what Iīd think. Can anyone give me an explanation for this, and what shall I try to solve the problem...?
In some plates everything works well, and in others not, so itīs not a permanent problem, but I test 400 samples in a run so itīd be very important for me to find a solution.
thanks



Hi, I don’t actually know what HRM or Cp is (is this the same as Ct?) but, is there any chance it might be the instrument playing up? I know with real time instruments (I use ABI 7500) if some of the wells are dirty (in the actual machine) it can skew your results big time. It might be worth doing some maintenance work on the instrument...




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