We are experiencing some troubles with the analysis of some data from several genes in CFX Manager. We want to adjust the baseline from some target genes data, as it was automatically set as to take some noise data in early cycles as actual take off. Nevertheless, when we try to use the software tools to set baseline parameters, the new baseline does not seem to be in concordance with them.
Now we want to try using the raw fluorescence data in some alternative software(s) to set the baseline according to what is written about this in the literature (i.e. having the baseline depend on standard deviation of the fluorescence from the first cycles).
Can anyone suggest any (preferably free) software to which I could export my raw data to perform this analysis and find my Cqs?
It's hard to get data out of CFX Manager. Have you tried to change the number of cycles to analyze?
I completely understand the issue of wanting to set the baseline. I learned how to do qPCR on an ABI system which allowed the user a lot more control setting certain parameters. It took me awhile to figure it out, but you can in fact set the baseline and cycle threshold in CFX Manager. Go to settings, then Baseline Threshold. In the new window that opens click User Defined and then you can adjust the baseline for each well according to which cycles you want the baseline to be between.
The way you describe setting the baseline "i.e. having the baseline depend on standard deviation of the fluorescence from the first cycles" it also sounds like you want to manually adjust the cycle threshold. To do this you need to change the analysis mode to target instead of fluorophore. Your should now be able to adjust the cycle threshold for each of your targets independently. To get the cycle threshold value go to settings, then Baseline Threshold. In the new window that opens go down to where it says Single Threshold. There you will see an option for auto calculated or user defined. Make sure user defined is checked and then the value that is displayed is the cycle threshold you have set according to how you want.
Hope this helps!
- Badges
- Science topic