Dear Sami, 

Actually the geNORM is an old software for gene expression normalization and analysis, while now we use the newer software of qBASE (the disadvantage here is of course that now one needs to buy it and it's not free any longer) in parallel to LiRegPCR (free software which you can download online http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR). 

But coming to your question to use geNorm, you run your reference genes and your gene of interest in the RNA concentration you found to have the best efficiency. Nevertheless, for each run, you need, in a way, standards, to run in paralleled so you can extract from your standard for each run the efficiency of the particular PCR. You can do it for example if you have plasmids with your HKG and gene of interest and then run this in series of dilutions to get a standard curve for each run. Than you can use the efficiency in a general way for the specific plate you have run to calculate the Q values (Q= E^(min Ct - sample Ct). But of course it assumes that all of your samples run with the same PCR efficiency.

But what you can do now is use the LinRegPCR program, and calculate with each for each well its own PCR efficiency. Then, if you still use the geNORM you will use the efficiency as the equation above to calculate for each sample its Q value using the minimum Cq value of all the runs for the specific HKG or your gene of interest, and the Cq value of the specific sample you wish to calculate the Q value for.

I hope this was in a way somewhat helpful for you and I wish you good luck.

I assume you have two different questions: 1) how to choose your HKG and 2) how to analyze the HKG using the efficiency of the PCR.

For the second question, I would recommend you to use the LinRegPCR first to get for each well run in your PCR its PCR efficiency and than use it in qBASE for the analysis of your HKG.

For the first question, you will get the resuts in the qBASE for ranking the HKG in most stable and least stable, so you will obviously choose the most stable one.

Dear Sami,

I guess that means you have a qBase+ licence in which case you can get support here.

https://www.biogazelle.com/qbaseplus/support

There's a manual with info on the reference gene validation on page 46 but if that is not clear enough, I've found that the Biogazelle Tech support is pretty good.

Unfortunately, my licence expired so I can't lead you through it step by step.