Using ABI system for qPCR and LinReg tool for analysis. When determining baseline manually it appears this error message in some points: "Invalid floating point operation" and stops the program. I realized that part of this error message is due to the NTCs, so I removed it from the raw data. But still appearing the same error message in other points...then I cannot keep going with the analysis. If I remove the points where appears the error message then the program run. But has anyone experienced this and have another option to solve the problem without remove the points from the raw file? Thanks!
Hi! It's a problem because you might be using some data with a corrected baseline (Rn or delta Rn).
Try to export separately the data from Sybr and for Rox (if you use Rox). You'll find this by exporting "component". Take only the values from cycle 1 to 40 (after it's the dissociation protocol). Then, devide each sybr value by the corresponding rox value. All values have to be positive (of >0 or you will get "invalid floating point operation"). Make the analysis on this data. If you're still stuck in this problem, I can give you a template excel file.
Which is the raw file that you're importing? Are you sure you're not importing a file that already has a baseline correction? I generally import the deltaRN file into LinReg from ABI.
Thank you Samantha! The raw file is the one that I exported from the SDS 2.4 and it is not corrected yet...
Hi! It's a problem because you might be using some data with a corrected baseline (Rn or delta Rn).
Try to export separately the data from Sybr and for Rox (if you use Rox). You'll find this by exporting "component". Take only the values from cycle 1 to 40 (after it's the dissociation protocol). Then, devide each sybr value by the corresponding rox value. All values have to be positive (of >0 or you will get "invalid floating point operation"). Make the analysis on this data. If you're still stuck in this problem, I can give you a template excel file.
Arnaud - you made a good point! the Rn (not deltaRn) is the sheet that should be exported into the LinRegPCR. Do you know if there's an easy way to export just the Rn values (instead of manually having to go through alter the spreadsheet?)?
You're welcome.
Samantha, we have an ABI 7300 qPCR system, and the only things I found to export are Delta Rn (but there are negative values), or component (and so you need to make a division by ROX values). Sorry, I don't have no better trick... I join to this message an Excel file as an example. Pleased to help you.
I don't know if it will be helpful. But the way that I export the data to run in the LinReg is: export the clipped file from the SDS 2.4, open it on excel and from the excel I import it into the LinReg. But I do not use the Delta Rn. I use the Rn values to correct the baseline. The problem that I mentioned before is that I had some Rn values less than 0, and very low expressed genes so I had to corrected the baseline manually instead of determining automatically the baseline with the LinReg using the Rn values.
Im using ABI FAST 7500 i try to import the exel file exported from the software (abi 7500) but i couldnt import any of the file into LinRegPCR. As if im using "Multicomponent data" which has well, cycle and SYBR, ROX. i have some negative SYBR values. What should i do? and how to import the data? my aim is to do inter-run calibration as my samples cant fit all in one 96 wells and i have 6 wells (2 re-run samples-3 replicates each) as a control for normalization.
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