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

TLDR

Real‑time PCR commonly normalizes RNA expression with housekeeping genes, but their variability across samples has raised concerns. The authors quantified 13 candidate housekeeping genes by RT‑PCR in peripheral blood mononuclear cell cultures and whole blood from healthy volunteers and tuberculosis patients, selecting genes from conventional panels and those previously reported as invariant in T‑cell culture. None of the commonly used housekeeping genes, including GAPDH, exhibited stable expression (variability >30‑fold), and genes previously deemed invariant in T‑cell culture also varied >34‑fold; only the human acidic ribosomal protein gene showed consistent expression and is recommended for normalizing mRNA in pulmonary tuberculosis.

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.