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Clinical Real-time PCR assay


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#1 tea

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Posted 10 December 2010 - 09:18 PM

Hello, I'm new to this forum and to RT-time PCR.

I have been given the job to validate a real time PCR assay (4 singleplex reactions with sybr green).

The conditions of the reactions are per manufacturer's recommendations. I performed a standard curve analysis and here are the results:

1st primer set (amplicon size: 600 bp) - slope: -3.79 (CT for 1x template is 20)
2nd primer set (amplicon size: 570 bp) - slope: -3.48 (CT for 1x template is 26)
3rd primer set (amplicon size: 612 bp) - slope: -3.2 (CT for 1x template is 20)
4th primer set (amplicon size: 537 bp) - slope: -3.63 (CT for 1x template is 21)

My questions are as follows:

1) I'm going to keep the 3rd primer set. What about the other primer sets, should I try to optimize them or redesign them?
2) I read somewhere that if the slope is higher than -3.6 (ideal is -3.3), then I should look for other primer sets.
3) For primer set 2, the slope is within range, but the CT is not perfect, should I try to optimize this reaction, and if so, how (increasing Mg?)

I really appreciate your help. Any comments or suggestions are welcome. Thanks!

#2 dtae

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Posted 30 December 2010 - 03:45 PM

The slope gives you an estimate of the PCR efficiency. Efficiencies over 100% (more than doubling every cycle) suggests contamination. Less than 100% suggests the reaction is working less well with each cycle. However, efficiencies estimated from the slope of a standard curve are also changed by inhibitors/enhancers in the standard curve dilution.
Efficiencies can also be determined at the individual reaction level using programs such as LinRegPCR.




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