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Threshold values

Threshold values - Real-Time PCR and Quantitative PCR Forum

Threshold values - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 11-03-2010, 09:55 AM
Pipette Filler
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Default Threshold values



Hi,

A quick question - I am running a basic qPCR experiment and analysing by the delta ct method (comparing target gene to endo control). However, I am wondering about the threshold level settings. By default, the software sets the threshold value at 0.2 in both cases. However, this is not an ideal threshold. When I click 'auto' threshold, they do change, but the values are different for my target gene versus my control gene. Does this discrepancy in thresholds matter? I know it would affect the final delta Ct value so am I better manually setting or letting the auto function do it's thing?

Thanks a million
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Old 01-12-2011, 01:34 AM
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Default Re: Threshold values

Did you ever decide whether using manual or auto threshold settings was better?

Should you use the same threshold for the same primer sets in different PCR runs? Or adjust it for each PCR? I'm having difficulties getting the same Ct values for the same samples in different runs.
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Old 10-26-2012, 10:33 AM
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Default Re: Threshold values

Contacted an AB rep about this and they recommend using the Auto Ct value. It's apparently the most consistent way to assign Ct thresholds on different samples, as the calculation is always the same and is based on slope etc.
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