Help with definitons for qPCR - (May/24/2015 )

Hello,

I recently did a qPCR (just a trial) using a ABI stepone plus system and i am stuck with the results. I am unable to understand the following:

1) Ct Mean: I have only one Ct value for a sample but it gives me a mean Ct mean value. How?

Even some samples went undetermined but it still shows a Ct mean value. Why?

2) What is meant by quantity and why is that empty?

3) What is Ct Threshold?

4) I have three Tm values. I assume that is the melting temp. of the primers. Am i correct? Why are there 3 Tm values?

Please find attached a screenshot of the result.

Thanks :)

In short all samples appeared negative, so even if the system reports a mean etc, they are undetermined.

1) Even with one single replicate, the system will still make the sum of all replicates (just the one) and divide by their number (1) to give you that mean. But the system correctly does not display a standard deviation for that sample.

2) I suppose you had a standard curve; quantity is empty because the Cqs of all samples fall beyond the standard curve.

3) The Ct (Cq) Threshold is the amount of fluorescence at which the instrument will determine a sample as amplifying. The instrument will assign a Ct to the sample based on the time where this value is reached.

4) You actually only have one Tm value. There are more columns for Tm values, but you only have one column filled. You can see however, that it's very variable - your data just looks negative.

One thing you should note, is that the instrument determined an auto baseline from cycle 3 to cycle 39 (that is, it determined to have measured only background fluorescence noise up to cycle 39).