Reference gene identification for normalisation of RT-qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model

Abstract

Objective: In quantitative reverse transcription-polymerase chain reaction (RT-qPCR) studies, the selection and validation of reference genes are crucial for the accurate analysis of MicroRNAs (miRNAs) expression. In this work, the optimal reference genes for RT-qPCR normalisation in plasma samples of rat middle cerebral artery occlusion (MCAO) models were identified.

Methods: Six rat MCAO models were established. Blood samples were collected before modelling and approximately 16-24 h after modelling. Two commonly used reference genes (U6 and 5S) and three miRNAs (miR-24, miR-122 and miR-9a) were selected as candidate reference genes, and the expression of these genes was detected with RT-qPCR. The acquired data were analysed using geNorm, Normfinder, BestKeeper, RefFinder and comparative delta threshold cycle statistical models.

Results: The analysed results consistently showed that miR-24 was the most stably expressed reference gene. The 'optimal combination' calculated by geNorm was miR-24, U6 and5S. The expression level of the target gene miR124 was similar when the most stable reference gene miR-24 or the 'optimal combination' was used as a reference gene. However, compared with miR24 or the 'optimal combination', the less stable reference genes influenced the fold change and the data accuracy with a large standard deviation.

Conclusion: These results confirmed the importance of selecting suitable reference genes for normalisation to obtain reliable results in RT-qPCR studies and demonstrated that the identified reference gene miR-24 or the 'optimal combination' could be used as an internal control for gene expression analysis in the rat MCAO model.

Keywords: RT-qPCR; circulating miRNA; middle cerebral artery occlusion; reference gene.