What are the basic calculations in qbase+?
qbase+ uses a generalized model of the delta-delta-Ct approach, thereby supporting the use of gene specific amplification efficiencies and normalization with multiple reference genes. All formulas of this model are detailed in Hellemans et al., Genome Biology, 2007.
Conversion of Cq values into relative quantities (RQs)
- Calculation of the average Cq value for PCR replicates
- The average Cq value is transformed into the RQ using the average Cq across all samples for that given gene as a reference point, and a calculated or user defined amplification efficiency
Conversion of RQ values into normalized relative quantities (NRQs)
- Calculation of the normalization factor (NF) for each sample is typically based on the RQs of the reference genes for that sample. Alternative normalization methods are available.
- The NRQ is calculated by dividing the RQ by the sample specific NF.
Conversion of NRQ values into CNRQs (calibrated normalized relative quantities)
Only needed when expression levels for a given gene are compared between samples measured in different runs.
Inter-run calibration is based on comparison of results for samples that have been measured for the same gene in different plates, i.e. inter-run calibrators (IRC):
- Calculation of the calibration factor (CF) for a given gene in a run based on the NRQs of the inter-run calibrators (IRCs) in that run.
- The CNRQ is calculated by dividing the NRQ by the CF
Final CNRQ or NRQ results can then be rescaled according to various user preferences. This only changes the scale of the data, but not the fold changes between the samples.
By default, expression levels are scaled to the average across all samples. Alternatively, expression levels can be rescaled to the sample with the lowest or highest expression, to a specific sample (e.g. untreated control), to the average of a certain group (e.g. all control samples), etc.