Boris you can find the documents that I made modeled after the equations in Hellemans paper on my Puget Sound Science Core Facility website: http://www.pugetsound.edu/academics/departments-and-programs/undergraduate/biology/science-core-facility/user-guides/ Just scroll down to the Bio-Rad CFX96 qPCR section. Let me know if you have any problems accessing them!

Here you will find how to do that:

http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf

Thank for your advice José,

but unfortunatelly I realised that I have different efficiency of target and reference gene, so I need to use Pfaffl equation and there It is problem with errors. Do you know how can I count efficiency of control using Pfaffl?

Thank you

no efficiency, but errors :)...Do you know how can I count error for control using Pfaffl?

Thanks a lot :)

Hi Boris

Difficult task here. I always use set of primers with very similar efficiencies and the delta Ct method, so I can do the error propagation as in the Applied Biosystems manual. But for Pfaffl method, calculations are not easy. The same author recommends the REST software.

Take a look at this document and you will get a better idea:

http://qpcrsymposium.com/presentations08/pfaffl.pdf

I couldn´t find any example on how to do the calculations in the papers by this author and others.

The way you are doing calculation, control will not have any error bar and in graph you have to present it as 1 without error bars. The reason is whatever the original value of your control is, your software is taking is a 1 and comparing it with others. Therefore, control will come without error bar and it is fine.

I recently had to hand calculate my expression values from the Ct values just as you describe. It took me awhile to track down all of the necessary equations, but I found the following document from GeNorm to be particularly helpful http://medgen.ugent.be/~jvdesomp/genorm/geNorm_manual.pdf. If you still need more information I'd be happy to share the hand calculation user guide and Excel template I made to calculate these values.

Thank you Amy a lot. I look at this document and try to count it and I let you know how it is going. :)

Hello Amy,

I looked at that document, but I am still a bit confused with that equations and also It doesn´t work in Excell 2007 and higher. Could you please send me a template? Thank you :)

All our formulas for error propagation during qPCR data-analysis are mentioned in attached paper. The formulas are integrated in Biogazelle's qbase+ software (http://www.qbaseplus.com). I recommend to analyse all samples at the same time in the software. qbase+ is compatible with LC480 output.

Article Hellemans J, Mortier GR, De Paepe A, Speleman F, Vandesompel...

Boris you can find the documents that I made modeled after the equations in Hellemans paper on my Puget Sound Science Core Facility website: http://www.pugetsound.edu/academics/departments-and-programs/undergraduate/biology/science-core-facility/user-guides/ Just scroll down to the Bio-Rad CFX96 qPCR section. Let me know if you have any problems accessing them!

Amy, thank you very much for the template. Finally, I have error bar for control sample counted by Pfaffl formula. Really helpfull and clear/understandable template. thanks a lot :) You saved my time and I don´t worry about it anymore.

Boris, you're quite welcome. I'm glad you found it useful!