Validation of housekeeping genes for normalizing RNA expression in real-time PCR

Abstract

Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.

Normalizing RNA Expression

Normalizing RNA levels to internal or housekeeping reference standards is widely used to control for sample error when measuring RNA expression levels. Choice of an appropriate reference standard is critical to avoid misinterpretations of results, and reference validation is crucial in assessing the utility of a particular experimental model. Dheda et al. (p. 112) report a validation protocol to identify the most suitable housekeeping standards in studies of pulmonary tuberculosis. They used real-time reverse transcription PCR (RT-PCR) to study the levels of 13 housekeeping genes expressed in blood cell cultures of groups of both healthy and tuberculosis patients. Only one gene, HuPO, was found to be suitable for normalization of RNA levels for these studies, unlike GADPH and β-actin that were each found to be highly variable. Their results show that housekeeping genes can be variable and prone to shifts in expression induced by experimental conditions, thereby causing problems for reliable normalization. In order to avoid significant inaccuracies when interpreting RNA expression data, this study emphasizes the importance of validating controls for individual experimental systems.

Acknowledgments

This work was supported by a grant from the British Lung Foundation.