How do I analyse a mixture of technical and biological replicates?
The most effective way of checking technical stability in Progenesis is to run technical replicates of a pool collected from all of your samples in an experiment. The pooled sample should be run at regular intervals throughout your data collection in order to monitor condition of the column, sample degradation and other factors that might affect analysis. You can make use of these technical replicate runs in Progenesis by setting up multiple experiment designs:
- Set up your experiment as normal, importing all of your runs (including your technical replicate runs of the pooled sample)
- Align your runs' retention times
- Set up a pair of experiment designs:
- For your first design, select Between-subject design; group your biological replicates and leave out the technical replicate runs of the pooled sample
- For your second design, again select Between-subject design, but this time include only your pooled sample runs, grouping them as a single condition
- Complete the rest of the workflow
You can then check the CV in the Max CV% column at Review Peak Picking (as well as in exported peptide measurements) for the technical replicate pooled sample runs for peptides you are saying are significant. You can cross reference these values with the CV in your true experiment design containing your biological replicates — for significant peptides you should expect the technical variance to be low compared to biological variance.
If you’ve already run your samples and have run technical replicates for each of your samples, you might want to refer to our blog post on analysing technical and biological replicates without pooled sample runs.