Which input data can I use for NormFinder?
The input data for Normfinder is supposed to be on a linear scale.
Thus, Ct-values (cycle treshold) from a real time RT-PCR should not be used directly.
Transform your Ct values to linear scale expression quantities (without any negative values) by using a standard curve or for assays amplifying at ~100% efficiency by 2-Ct.
The NormFinder software log(N) transforms your data when you upload them, if they are not log-transformed already.
Don't use delta-Ct as input data.
How do I calculate the delta-Ct for my genes of interest by using a combination of normalization genes?
If a combination of two or more genes gives the lowest stability value, simply average the raw Ct values of these genes for each sample, and use this value for further calculations.
What is the Normalization factor NF?
The Normalization factor is typically calculated as the geometric mean of the reference genes included.
How do I deal with replicates in my data sheet?
NormFinder aims at identifying one or more genes that are stably expressed across your biological samples.
Technical replicates can be treated as one measurement of the same sample, calculate the median for use in NormFinder.
Biological replicates should be treated as individual samples.
Different time points and independent treatments which infer expression variation should be considered as grouping variables.
NormFinder works with three and more groups, you will obtain an inter-group variation value for each possible pair of groups.
How do I download and install the .xla MS Excel add-in?
In Office 2007 and 2010 the .xla file will be saved with a .xls file extension. Simply install the add-in by double-clicking and enabling macros and data connections. You will find a new tab Add-ins with the NormFinder software.
If you install the file by browsing to it under the menu Excel-Tools-Add-ins, you need to chose "All File Types" since the .xls file will not be shown.
In Office 2013 and 2016 download and double-click the .xla addon. Allow this macro to run and click "Activate". You find the Normfinder-Add-in under the add-ons tab.
I need to calculate the "accumulated standard deviation" from Normfinder - can the software do this?
Unfortunately - no. There are no plans for releasing new versions of NormFinder with this functionality.