Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage

Abstract

Background: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate reference gene selection. Reference genes should be stably expressed across different experimental conditions, however, transcript levels of common reference genes have been shown to vary considerably. Reference gene panels have therefore been proposed to overcome this potential confounder. Results: The present study evaluated the stability of seven candidate reference genes in the striatum and overlying cortex of collagenase-induced ICH in rodents at survival times of 5 and 24 hours. Transcript levels of the candidate reference genes were quantified and ranked in order of stability using geNorm. When our gene of interest, transient receptor potential melastatin 2 (TRPM2), was normalised against each reference gene individually, TRPM2 mRNA levels were highly variable. When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats. Conclusion: The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH.