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Reference Gene qPCR


  • Determine which of your reference genes you are going to normalize to.

Why am I doing this?

Reference genes need to be stable across your control and experimental samples in order to be useful. If expression between the two differs, you will be normalizing to two different values. Reference genes should always be validated before you use them in your experiments.

Typical plate setup for three candidate reference genes with three technical replicates for each biological sample:


*C1 = Condition 1, C2 = Condition 2, and BR# = Biological Replicate

Template Material

Note: Once you've diluted all of your cDNA samples store them at 4°C to avoid complications with freeze/thaw

What you are looking for

  • Technical replicates with a standard deviation below 0.2. This confirms the accuracy of your results.


There is no detailed analysis for this step. Look for references that match the "what you are looking for" criteria and select:

  • At least 2 reference primer sets that show no significant difference between control and experimental conditions. The standard deviation of the Cqs for all biological replicates should be low, preferrably less than 0.5.

If you are seeing differences between replicates and conditions, it is likely due to technical issues encountered when setting up the qPCR plate. The first few times you run qPCR the results can be messy, and sometimes you have to redo things to get accurate results. That being said, if you're a qPCR pro, or if you keep getting the same results over and over you may have to design primers for a new reference.

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Contributors to this topic Edit topic KateElston, JuliePerreau
Topic revision: r6 - 2022-07-19 - 22:56:58 - Main.KateElston
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