Using ribosomal protein genes as reference: A tale of caution

Abstract

Background: Housekeeping genes are needed in every tissue as their expression is required for survival, integrity or duplication of every cell. Housekeeping genes commonly have been used as reference genest to normalize gene expression data, the underlying assumption being that they are expressed in every cell type at approximately the same level. Often, the terms "reference genes" and "housekeeping genes" are used interchangeable. In this paper, we would like to distinguish between these terms. Consensus is growing that housekeeping genes which have traditionally been used to normalize genes expression data are not good reference genes. Recently, ribosomal protein genes have been suggested as reference genes based on a meta analysis of publicly available microarray data. Metholodogy/Principal Findings: We have applied several statistical tools on a dataset of 70 microarrays representing 22 different tissues, to asses and visualize expression stability of ribosomal protein genes. We confirmed the housekeeping status of these genes, but further estimated expression stability across tissues in order to assess their potential as reference genes. One- and two-way ANOVA revealed that all ribosomal protein genes have significant expression variation across tissues and exhibit-dependent expression behavior as a group. Via multidimensional unfolding analysis, we visualized this tissue dependency. In addition, we explored mechanisms that may cause tissue dependent effects if individual ribosomal protein genes. Conclusions/Significance: Here we provide statistical and biological evidence that ribosomal protein genes exhibit important tissue-dependent variation in mRNA expression. Though these genes are most stably expressed of all investigated genes in a meta-analysis they cannot be considered true references genes. © 2008 Thorrez et al.