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

Heleen Nailis,Hans J Nelis,Tom Coenye,Dieter Deforce,Filip Van Nieuwerburgh

doi:10.1186/1471-2199-7-25

Abstract

BackgroundCandida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts.ResultsThe 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.ConclusionIn 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.

Highlights

Candida albicans biofilms are commonly found on indwelling medical devices
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

Summary

Introduction

Candida albicans biofilms are commonly found on indwelling medical devices. the molecular basis of biofilm formation and development is not completely understood. 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 [1,2,3]. Cells released from these biofilms can migrate to the bloodstream and can cause systemic infections [4,5]. The molecular basis of C. albicans biofilm formation and development is not completely understood It is, well-established that interaction of C. albicans with host cells or inert surfaces leads to changes in gene expression. The ALS3 gene, another gene belonging to the ALS gene family has been shown to be upregulated in C. albicans hyphae, which suggests that ALS3 might play a role in biofilm development by this organism [15]

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Discussion

Conclusion