Normalization aims to account for systematic technical sources of variation so that biologically-driven changes in gene expression can be better detected between samples. There are two options for normalization in EcoToxXplorer: delta-Ct and quantile.
-
Delta-Ct is the most commonly used normalization for qPCR data, and so we suggest starting with this method for differential expression analysis.
-
Quantile normalization is becoming more popular as qPCR arrays get larger. Also, if the housekeeping genes are not stable across conditions, quantile normalization can still be used since it does not use the housekeeping genes as internal references. Quantile normalization is the only option for dose-response modeling because the curve fitting algorithm requires only positive values, and delta-Ct produces both positive and negative values.