Jornal de Biometria e Bioestatística

Ratios and Housekeeper Normalization

Abstract

Justin R Brown and Valentin Dinu

A common practice in gene expression studies is to use ‘housekeepers’, i.e., genes expected to be expressed at relatively constant levels across experimental conditions, to normalize data. The process is to divide an expression value by some composite of one or more stable housekeepers to remove the effect of processing and nuance variables. Despite its reverence and widespread use, we argue that this approach is fundamentally flawed on multiple levels. The outcome of housekeeper normalization is a set of ratio variables which are not amenable to many standard statistical tests. There are no universal housekeeper genes and even within specific cohorts proposed housekeeper genes often fail to replicate. Furthermore, there is also no single agreed upon algorithm for performing housekeeper normalization or agreement regarding what constitutes a good housekeeper. We urge researchers to consider the use of alternative methodologies in their research.