Using GeNorm for determing stable reference gene

Hello everyone,

I am posting after a long time. Today, I wanted to explain the GeNorm method for calculation of stable reference genes. Maybe some of you already know how to calculate it but yes there maybe someone like me who didn’t quite understand how to calculate and input the data 😉

With the MIQE guidelines in action, most researchers are now using multiple reference genes for normalization of their gene of interest (GOI). But to use more than one reference genes you need to validate the genes before use. There are many open source plugins and software available to validate reference genes. One of the most popular being the GeNorm with more than 7000 citations. It was developed by Jo Vandesompele way back in 2002.

To begin with GeNorm, you need to convert your raw Cq values to relative quantities before input to GeNorm excel sheet. For example, consider the following Cq values from biological replicates A and B:

BIOLOGICAL REPLICATE A1BIOLOGICAL REPLICATE B

2Now find the minimum value from these Cq values using Excel function “MIN.” Then subtract the “MIN value” from all other values by using the formula 2(Min value – Cq value). This will give values in a relative form with the highest ∆ct value as 1, and all other values are less than 1.

Now, you need to prepare the obtained values for input to GeNorm. For this, you need to have an excel sheet (2003-2007 version). Leave the first cell blank i.e. cell A1 should be empty. Fill in the gene names in the rows i.e. A2, A3, A4….. and write in the sample name in the columns i.e. B2, B3, B4,B5…. Save the file as Excel sheet 2003-2007 (.xls).

Your input data would look like this:

3Time to open GeNorm (Excel sheet): and it would look like this. Click on Start GeNorm.

4This would be followed by a dialog box (not shown). After you close the dialog box a blank page (similar to the picture below) would appear.

5Click on “Input data manually.”

6A new dialog box would appear which would ask you to define the no. of rows and columns you need.

7Irrespective of the no. of rows and columns you chose, GeNorm would give only two columns. So, you have to enter the no. of columns manually.

8Now, click on load input data:

9Once the data has been loaded, go to GeNorm and click on automated analysis:

10This would generate the first chart, which shows the least stable genes (on the left) and most stable gene (on the right).

11Clicking on the graph icon would generate the second chart:

12Which would be like this:

13The second table shows the pairwise variation V between two sequential normalization factors containing an increasing number of genes. The proposed cut-off value is 0.15.

That’s all from GeNorm. Now, you know which are the genes that you can use as reference genes and how many do you need to have accurate data. In my next posts, I would explain how to use Normfinder and Bestkeeper. Until then, good bye and happy data analyzation. J

REFERENCE:

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2 thoughts on “Using GeNorm for determing stable reference gene

  1. Shashank Patole

    Hi Addictive Brain.
    Thanks for the post; it really put things into clearer perspective. I have a small confusion though. Does a separate minimum have to be obtained for each biological replicate belonging to the same target, or does the minimum have to be obtained from all biological replicates for that target?

    Like

    Reply

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