Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

Carme Gubern,Jesús R Morales,Víctor G Romera,Olivia Hurtado,Joaquín Serena,María A Moro,Judith Mallolas,Rocío Rodríguez,Ignacio Lizasoain

doi:10.1186/1471-2199-10-57

Abstract

BackgroundStudies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha) to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models.ResultsThe expression stability of the candidate reference genes was evaluated using the 2-ΔC'T method and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes.ConclusionWe have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.

Highlights

Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets
Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup
Our results show that Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), Ribosomal Protein L13A (Rpl13a) and Peptidyl-prolyl cis-trans isomerasa (Ppia) together with other candidate genes, namely β2m, Glucoronidase B (Gusb), 18S rRNA and Ornithine decarboxylase antizyme 1 (Oaz1), are not appropriate as housekeeping genes for the permanent MCAO (pMCAO) model

Summary

Introduction

Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used. The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha) to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models. The ideal housekeeping gene for qRT-PCR would be one whose mRNA is consistently expressed at the same level in all samples under investigation, regardless of tissue type, disease state, medication or experimental conditions, and would have expression levels comparable to that of the target [14,15]. Careful evaluation and validation of control genes is required prior to using them in each individual case to avoid possible inaccuracies stemming from the use of an unsuitable reference gene [14,16,18,19,20]

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