Removal of Between-Plate Variation in qPCR with Factor Correction: Completion of the Analysis Pipeline Supported by RDML

Abstract
Quantitative PCR is the method of choice in gene expression analysis. However, the number of experimental conditions, target genes and technical replicates quickly exceeds the capacity of the qPCR machines. Statistical analysis of the resulting data then requires the correction of between-plate variation. Application of calibrator samples, with replicate measurements distributed over the plates assumes a multiplicative difference between plates. However, random and technical errors in these calibrators will propagate to all samples on the plate. To avoid this effect, the systematic bias can better be corrected when there is a maximal overlap between plates using Factor Correction [Ruijter et al. Retrovirology, 2006]. The original Factor Correction program is based on Excel input and calculates corrected target quantities. To implement this correction into the analysis pipeline from raw data through LinRegPCR into qbase-plus, a new version of the program was created to handle RDML files. This version saves the corrected N0 values as efficiency-corrected Cq values to be used in further calculations. This program thus completes the analysis pipeline of qPCR data supported by RDML.