Ct variation/Ct limit/r² values/efficiency errors

[wozzels]

Hey guys, I have some important questions in order to accurately evaluate some real-time data from students in my lab. Here they go:

If analyzing real-time pcr data, I was wondering about these important points:

1) Suppose you have triplicates for each sample/standard: the Ct-values of these three replicates are never exactly the same, but which degree of variation is allowed and should you remove wells of which the Ct-value is very different from the other 2? Is the cut-off around Ct +/- 0.15? And are data with Ct +/- 1.0 reliable or not at all?

2) If you read data or have wells which have Ct-values of more than 35, are these data or wells usable or is 35 the maximum allowed Ct-value? (I thought more than 35 was comparable to the baseline threshold)

3)When real-time data have correlation coefficients (r²) of 0.91-0.95, are these data reliable? (I thought data should have at least 0.98 r²)

4) If you have data with efficiencies of more than 100 % (standard curve slope > -3.33, for example -2.90), are these data reliable? Is the > 100 % efficiency due to improper settings of the threshold/baseline or due to pipetting mistakes?

If you have answers to any of these questions, that would be extremely helpful!

Thank you in advance and greetings!!!
Wozzels