Contents - Index


NormFinder

 

NormFinder is an algorithm that attempts to find the optimum reference genes out of a group of candidate genes. It can also, in contrast to geNorm, take information of groupings of samples into account, such as untreated/treatment1/treatment2, sick/healthy, or different stains. The result is an optimum (pair of) reference gene(s). The resulting pair might have compensating expression, so that one gene e.g. is slightly overexpressed in one group, but the other gene is correspondingly underexpressed in the same group (Andersen et al. (2004), Cancer Research 64 5245-5250). 

 

Open the Ref Gene tab among the analyses tabs in the top of the main window, and press the NormFinder button to load the analysis into the Control panel. If there are classification columns in the active data file, there will be an drop-down list in the Control panel where you can select a group defining column. It is not visible if there are no classification columns. You need at least three genes to run the analysis, and the data cannot contain any negative values, so make sure that it is not mean centered or autoscaled

 

    

 

One group

When all samples belong to the same group, and there are no classification columns in the data file, there is no drop-down list in the Control panel. If you have classification columns for other purposes than to define groups, select Ignore groups in the Classification column drop-down list. 

 

NormFinder is applied on data in logarithmic scale such as Cq values or fold changes. If the data is on linear scale, tick the Apply log2 check box, and GenEx will convert the data to logarithmic scale only for the NormFinder analysis. The result is summarized in the Control panel where you can see the optimal reference gene(, as well as its variability. By ticking the other check boxes in the Control panel, you can control what other output you want.

 

    

 

If the View plot check box is ticked, a bar plot is generated with one bar per gene representing the gene variability. The optimum reference gene is highlighted in red.

 

    

 

If the View stability matrix check box is ticked, you get to see a table containing each genes standard deviation (SD) as well as the accumulated standard deviation (Acc. SD) when more than one reference gene is chosen. The accumulated standard deviation is calculated with the following formula,

    

In the right side of the table is the suggested reference gene(s) and its/theirs standard deviation. The accumulated standard deviation is a great indicator of the optimal number of reference genes. It is plotted against number of reference genes in a line plot where the optimal number of reference genes is indicated in red.  

 

    

 

    

 

Multiple groups

If the samples belong to different groups, such as treated and untreated, Normfinder can separate between sample variability and bias between the groups. This requires that the data file has a classification column that specifies which group each sample belongs to. Select the group defining classification column in the drop-down list in the Control panel. NormFinder calculates both intragroup variance, describing the stability of the gene expressions within each group, as well as the intergroup variance, which describes the stability of the gene expressions between the groups. The result is the an optimum pair of reference genes. The resulting pair might have compensating expression, so that one gene e.g. i slightly overexpressed in one group, but the other gene is correspondingly underexpressed in the same group. Hence, the optimum pair may not include the optimum single gene, although usually it does. 

 

NormFinder is applied on data in logarithmic scale such as Cq values or fold changes. If the data is on linear scale, tick the Apply log2 check box, and GenEx will convert the data to logarithmic scale only for the NormFinder analysis. The result is summarized in the Control panel where you can see the optimal reference gene, the optimal pair of reference genes, as well as their variability. By ticking the other check boxes in the Control panel, you can control what other output you want.

 

    

 

If the View plot check box is ticked, a bar plot is generated with one bar per gene representing the gene variability. The optimum reference gene is highlighted in red, and the optimum pair in yellow. If the optimum gene is a part of the pair, there is only one yellow bar.

 

    

 

If the View stability matrix check box is ticked, you get to see a table containing each genes standard deviation (SD), and the suggested reference gene  and suggested gene pair, as well as their variability.

 

    

 

There are check boxes for viewing the Intragroup variation and Intergroup variation. If ticked, they display tables that show the different variances for each gene and group.

 

    

 

    

 

Watch the tutorial Find reference genes from one group on how to select optimum reference genes from one sample group, and the tutorial Find reference genes for multiple groups on how to select optimum reference genes from grouped samples. To learn more about NormFinder, visit www.mdl.dk/publicationsnormfinder.htm