How many reference genes do you use in qPCR?

In the past, we’ve discussed the importance of selecting appropriate reference genes for your qPCR experiment (also see point 7 of the MIQE guideline checklist). This means that it is important to select genes that do NOT exhibit any changes in expression under the treatment conditions you are studying. This is easier said than done!

“Once upon a time” everyone used either beta actin, 18s, or gapdh as reference genes. Their expression never changes, right? Wrong! So which genes should you choose? If you try to figure it out using previous papers, how do you know that they’ve chosen the correct genes? If you run a few genes side-by-side and try to compare their expression both under treatment and control, which one should you set as the baseline and which one can you say is for sure moving (it’s all relative isn’t it)?

One of my twitter friends told me that she uses six reference genes in her qPCR experiments. I used to use two. That got me thinking…how many reference genes does the “average” lab use? Please help satisfy my curiosity by participating in the poll below!

How many reference genes are you using in your qPCR experiment?

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3 Responses to “How many reference genes do you use in qPCR?”

  1. Jo Vandesompele says:

    Great question! I answered 2-3 reference genes as we most often use 3, but the correct answer would be “it depends”. The more variability in your experiment, the more reference genes you need. Two genes is an absolute minimum number in order to determine their expression stability. Sometimes, this is sufficient to normalize the experimentally induced variation away; sometimes you need more genes. I highly recommend to set up a pilot experiment in which you determine which and how many genes are required for accurate normalization. genorm is a great tool to help you with that process (

    Jo Vandesompele
    Ghent University, professor
    Biogazelle, co-founder

    • avi_wener says:

      Jo- Thanks for the comments. Great advice. One lesson is true to everything in life…it always “depends!” I’m just floored by how many people do qPCR without even thinking about this question or testing their assumptions or validity of their data.

  2. How Do you calculate results after using 2 or more reference genes? What method do you use? I used ddCt before but don’t know how to analize data with 2 reference genes.

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