I need to obtain the raw amplification data from the StepOne software in order to calculate the PCR amplification efficiency using the LinRegPCR software.
According to the developers of LinRegPCR, the data must be "corrected for the technical background and, if used, the internal reference fluorescence (e.g. ROX) but should NOT be corrected for the fluorescence baseline".
Thank you in advance.
Yes, you can do that!!!! I used Linreg to analyse amplification data from StepOnePlus for 2years now. I strongly advise you to properly define all targets (genes you're interested in - master mix with the relevant primers) and all samples IDs before or after the PCR run. Then, from the Analysis section, inspect the quality of the data and remove the points you don't want to analyse (for an example the no-amplification in the negative controls). Then press Analyze. Then choose Export and select Sample setup and Amplification data. This will generate an Excel file with two tabs- Sample Setup and Amplification data. Open the tab "Amplification data" and then open LinReg. For StepOnePlus without the VeriFlex option activated, I choose column A trough E and row 8 trough 3848. In any case, you should be sure that LinReg picks up the Rn value (which = fluorescence from Sybr/ Fluorescence from Rox) and NOT the delta Rn. Than play with LinReg so that you define your amplicon groups, determine baselines, etc. Once done, save the data as Excel. Then match the LinReg output file (this will appear as a third tab) with the Sample Setup tab.
In case you get weird messages from LinReg, check out the Multicomponent plot in the Analysis section in StepOnePlus.
For some unknown for me and for ABI technical support reasons, sometimes their instrument gives negative values for the Sybr fluorescence for the first several cycles before reaching the threshold which has no physical sense at all, but it could force you do correct baseline for each sample individually in LinReg, as LinReg treats negative Rn values as zero and cannot correctly determine the baseline automatically (and really pisses me off, but it's ABI instrument issue, not LinRegPCR bug).
Anyway, LinRegPCR is a great software and it helped me to analyze more then 200 qPCRs already! Read the manual and if you need some sample data sets, let me know.
Yes, you can do that!!!! I used Linreg to analyse amplification data from StepOnePlus for 2years now. I strongly advise you to properly define all targets (genes you're interested in - master mix with the relevant primers) and all samples IDs before or after the PCR run. Then, from the Analysis section, inspect the quality of the data and remove the points you don't want to analyse (for an example the no-amplification in the negative controls). Then press Analyze. Then choose Export and select Sample setup and Amplification data. This will generate an Excel file with two tabs- Sample Setup and Amplification data. Open the tab "Amplification data" and then open LinReg. For StepOnePlus without the VeriFlex option activated, I choose column A trough E and row 8 trough 3848. In any case, you should be sure that LinReg picks up the Rn value (which = fluorescence from Sybr/ Fluorescence from Rox) and NOT the delta Rn. Than play with LinReg so that you define your amplicon groups, determine baselines, etc. Once done, save the data as Excel. Then match the LinReg output file (this will appear as a third tab) with the Sample Setup tab.
In case you get weird messages from LinReg, check out the Multicomponent plot in the Analysis section in StepOnePlus.
For some unknown for me and for ABI technical support reasons, sometimes their instrument gives negative values for the Sybr fluorescence for the first several cycles before reaching the threshold which has no physical sense at all, but it could force you do correct baseline for each sample individually in LinReg, as LinReg treats negative Rn values as zero and cannot correctly determine the baseline automatically (and really pisses me off, but it's ABI instrument issue, not LinRegPCR bug).
Anyway, LinRegPCR is a great software and it helped me to analyze more then 200 qPCRs already! Read the manual and if you need some sample data sets, let me know.
Thank you so much Petar!
I noticed that I can also exclude the negative controls and unwanted samples directly on the LinRegPCR interface by checking the "Mark sample as excluded" box on the Individual Sample tab.
About the negative values that you mentioned, my data only shows negative values in the delta Rn column, which in this case I think it wouldn't be a problem, unless I am doing something wrong when I export the Amplification Data.
To be honest I only need LinRegPCR to calculate the PCR efficiencies, so I can include them in the Pfaffel's equation.
Tiago, I use LinReg for the very same purpose and it works really great! Lucky you don't have negative Rn :)
I independently checked if the "real" efficiency (by dilution curve) is the same as the calculated by LinReg - well it is not in absolute value, but there is a strong correlation, so I trust it. Moreover this method has been published and later - reviewed.
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