T-test and RNA Spike-ins

[MicGriffin]

I have completed a paired t-test comparing two different treatments. However the RNA Spike-ins 2 and 4 are appearing on the top of the list as being significantly different. I have looked at the original list after the pre-analysis and there appears to be a only small differences (<1 Cq) between the spike-in values of the two treatments. Should I be observing spike-ins at the top of my t-test list in GenEx? In addition I am using normalisation by global mean and should the spike-ins be included for this?

[Mikael Kubista]

This does not have to be wrong, but caution is recommended. If the Cq´s of the spikes (after interplate calibration if needed) and normalization to the same total amounts of samples are congruent then procedure should be reproducible. If they then differ (statistically) after global normalization, it suggests that the overall expression under the two conditions differ. If you like a second opinion by a statistician contact us on: services@tataa.com.