Validation of reference genes for qPCR studies on Caco-2 cell differentiation.

Validation of reference gene expression stabilities is a prerequisite for reliable normalization of qPCR data. The present study assessed the variation of six reference genes (ACTB, GAPDH, B2M, HPRT1, SDHA, YWHAZ) in Caco-2 cells under the influence of different growth supports and cultivation periods. Genes were ranked according to their stability using the geNorm software. To verify the influence of reference gene selection, ALPI gene expression during differentiation was quantified using the most or the least stable reference gene for normalization. Experimental conditions significantly affected the expression levels of reference genes. Whereas GAPDH and ACTB were revealed as most stable genes, SDHA was the least stable one. The extent of ALPI gene expression was significantly changed by the selection of the reference gene. This study provides a basis for qPCR studies related to both the differentiation process of Caco-2 cells and the elucidation of cell behaviour influenced by surface modifications.