Abstract :
In dairy animals, gene expression analysis has become increasing key to understand the
biological processes occurring in mammary gland development that shape future milk
potential. Selecting high-stability reference genes is crucial to interpret real-time qPCR data.
This study investigated the expression stability of five top-ranked candidate reference genes
in the goat mammary gland through three assays comparing different experimental conditions
(physiological states, sample types and experimental treatments). The expression
stability of genes including ˇ-actin, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA,
cyclophilin A and ribosomal protein large P0 was analyzed. Normalization for each experimental
condition expression data revealed a different reference gene. Nevertheless, in our
various assays, genes encoding for ribosomal proteins, 18S rRNA and RPLP0 presented the
best expression stability. This result has been confirmed using a combined analysis of stability
on the three assays. All genes showed the same distribution within and among the
three assays and a different distribution between Ct variability and GeNorm normalization.
In addition, the application on Catenin B1 expression using an inappropriate reference
gene confirmed erroneous variations in interpretation. To conclude, there is no single ideal
reference gene for caprinemammarygland studies andwerecommend using a panel of topranked
reference genes, including RPLP0, at the beginning of each experiment to validate
the most stable(s) gene(s).
