Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (β-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase β-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase).Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours.In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours.Candida albicans is an important human fungal pathogen that is associated with biofilm formation on indwelling medical devices like urinary catheters, dental prostheses and silicone voice prostheses [1-3]. Cells released from these biofilms can migrate to the bloodstream and can cause systemic infections [4,5]. Fungal biofilms are highly resistant against most commonly used antimycotics [6]. Since no therapy is able to completely eradicate C. albicans biofilms, colonization of these medical devices often results in their functional loss and in most cases necessitates removal and/or replace