Hi there,
Has anyone successfully used GeNorm, one of the free programs to determine best reference gene(s) for qPCR? I cannot figure out how to format my qPCR data to get the qbase+ program to upload my data. If anyone can provide an example or description of how they formatted their files, I would hugely appreciate it.
Ellen Ingolfsland
I don't tend to use qbase, because...frankly I cannot afford it, but I do use the original genorm macro, and for that you need to convert your data to linear relative quantities (0-1) on a per-gene basis, using efficiency(lowest Cq - sample Cq) such that your sample with the highest expression has a value of 1, and everything else is fractions of that.
Then stick this data, for each gene, for each sample, into an excel table with the gene names along the top row, and the sample IDs down the leftmost column (top left corner will thus be blank).
Then run the genorm excel macro and import your data, hit calculate, and then the automated analysis (it will warn you that this will auto generate two graphs etc: this is good, this is what you want). You can then export the data this second step generates.
Ellen Ingolfsland To prepare qPCR data for GeNorm, do the following steps:
1. Make a spreadsheet with the raw Cq (quantification cycle) data for your potential reference genes and target gene. Each column should correspond to a particular gene, and each row to a single sample.
2. The spreadsheet should be saved as a tab-delimited text file (txt) or a comma-separated value (csv) file.
3. Launch the qbase+ program and navigate to the "GeNorm" tab.
4. Choose your text or csv file and click the "Import Data" button.
5. Follow the on-screen instructions to define the column headings and normalization factors.
6. When you've imported your data, click the "Calculate" button to compute the stability values for your potential reference genes.
7. Choose the most stable reference genes for normalization of your qPCR data using the GeNorm output.
It is crucial to note that GeNorm is only one of numerous programs that may be used to find the optimum reference genes for qPCR, and the particular processes for preparing data may differ depending on the application.
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