You can find the details on how to convert the targets in the Taking into account amplification efficiencies section of Analyzing gene expression data in qbase+
In Analyzing gene expression data in qbase+ we have already checked the stability of the reference genes (see Normalization section). We determined that Flexible did not show stable expression.
You can find the details on how to convert the targets in the Normalization section of Analyzing gene expression data in qbase+.
Appoint Sample01, Sample02 and Sample03 as IRCs.
Leave the Analysis wizard by clicking the Close wizard
button in the top menu.
Intermediate results
(red) in the Project ExplorerInterrun calibration
(green)This opens the Interrun calibration window:
New
button (blue) to create a IRCSample01
in the list of Other samplesAdd Sample
button (purple)Sample01_2
in the list of Other samples.Add Sample
button (purple)You have appointed the first IRC (grey), now do the same for the other two IRCs.
Remember that for each target the variability of the normalized expression levels of the IRCs between different runs will be used to adjust the other normalized expression levels of that target gene. The adjustment is done by amplifying the normalized expression levels with a calibration factor that is calculated based on the normalized expression levels of the IRCs. Since variability between runs is the same for each IRC, you expect that all IRCs measure the variability between the runs to the same extent, hence leading to similar calibration factors.
Open the Calibration Factors tab (red) of the Interrun calibration window and look at the result for Duvel:
You see that IRC2 returns a substantially different calibration factor in Run5 (green) so the validity of this IRC should be interpreted with care. For Leffe the IRCs also gives inconsistent results in Run5. Switch to the results for Leffe by selecting Leffe
in the Targets list (blue)
Open the target bar chart for Palm. You see that the pattern Palm showed in the first three runs (sample01 to sample16): high expression in the odd and low expression in the even samples is reversed in the samples from Run4 and Run5 (sample17 to sample25). In the latter runs you see high expression in the even and low expression in the odd samples. However, without annotation for Run4 and Run5 (which samples are treated and which not) it’s impossible to interpret the bar chart.