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Reference gene identification for normalisation of RT-qPCR analysis in plasma samples of the rat middle cerebral
Hui Zhou1, Xin Yang1, Jiayi Yu1
1Shanghai Innostar Bio-tech Co. Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China.
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.
