2 replies to this topic

[hgrombi]

Hello,
I have not much experience with LC480 but recently I´ve tried some duplex Real-time PCR on LC480. It´s a duplex reaction using FAM and HEX probes (using Color Compensation). After 2nd Derivative Max Analyses amplification curves of some samples (they should be negative) do not show any amplification. However, the software gives some Cp in the table. Could somebody advise me how is that possible and I could improve the data?
Thanks a lot.

Hana

[Trof]

Can you post the screenshot of the table/graph?
Is it really negative or just so low it looks negative, from the default graph setting? What is the color of the analysed curve, green (negative) or red?
What is your analysis setting "High specificity" or "High confidence"?

[leelee]

Are the cp values high? Does the software call them negative?

If they are high, it is possible that the software is calculating the Cp from the small increase in signal that you sometimes get at the very end of a run from probe degradation (say around cycle 35+).
Have a look at the individual curves and if necessary you can raise the threshold to exclude these samples and recalculate your Cp values.

I would also recommend contacting your local Roche tech rep if you need further help, the manual is in German English which is just about incomprehensible BUT the tech support is (at least I have found) exceptional