Setting Threshold for qPCR

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Pat101
Pat101's picture
Setting Threshold for qPCR

I normally let the machine set the threshold level for my qPCR reactions (ABI STepOne Real Time PCR Machine).  But I have noticed that these values can vary from 0.02 to 0.14 units for the same plasmid standards.  These values are within the 10 standard deviations suggested in the literature but as you can see this varies the quantitation because the Ct  values are affected.  How would you go about establishing a manual threshold value?

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Pat101, 

There really is no exact answer to your question since setting the threshold manually is subjective. Having said that, there are times when you have to do it, like when your signal is very high and you get amplification earlier than the 15 cycles (the instrument is designed to use cycles 3 through 15 as the baseline, and it is not smart enough to change this setting if signal appears earlier than cycle 15). What I have done in the past when I need to set the threshold level by hand is the following: start with the automatic threshold and start changing it in either direction. Every time, look at the slope and R2 that is calculated automatically. Chose the threshold that gives you the best slope and R2 for your assay (assuming that you are running a standard curve with your experiment). If you cannot do this, an alternative is to look at your amplification curves in log view. Simply put the threshold line in the middle of the upward sloping part of the curve; that should be the ideal threshold level. 

Good luck

yshin
yshin's picture
Would you say that you couldn

Would you say that you couldn't compare results from 2 runs if the threshold were different? Also, if the threshold using one primer/probe set is higher than another primer/probe set that detects the same gene, would you say that the one with the higher threshold is less sensitive/specific?