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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2022-09-01
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| Series: | Veterinary Medicine and Science |
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| Online Access: | https://doi.org/10.1002/vms3.879 |
| _version_ | 1828388092495527936 |
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| author | Hui Zhou Xin Yang Jiayi Yu Jingyi Xu Ruiwen Zhang Ting Zhang Xijie Wang Jing Ma |
| author_facet | Hui Zhou Xin Yang Jiayi Yu Jingyi Xu Ruiwen Zhang Ting Zhang Xijie Wang Jing Ma |
| author_sort | Hui Zhou |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T06:05:48Z |
| format | Article |
| id | doaj.art-d32e481790454f019877f88400cc0b97 |
| institution | Directory Open Access Journal |
| issn | 2053-1095 |
| language | English |
| last_indexed | 2024-12-10T06:05:48Z |
| publishDate | 2022-09-01 |
| publisher | Wiley |
| record_format | Article |
| series | Veterinary Medicine and Science |
| spelling | doaj.art-d32e481790454f019877f88400cc0b972022-12-22T01:59:42ZengWileyVeterinary Medicine and Science2053-10952022-09-01852076208510.1002/vms3.879Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion modelHui Zhou0Xin Yang1Jiayi Yu2Jingyi Xu3Ruiwen Zhang4Ting Zhang5Xijie Wang6Jing Ma7Shanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaShanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaShanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaShanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaShanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaShanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaShanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaShanghai Innostar Bio‐tech Co. Ltd. China State Institute of Pharmaceutical Industry Shanghai People's Republic of ChinaAbstract 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.https://doi.org/10.1002/vms3.879circulating miRNAmiddle cerebral artery occlusionreference geneRT‐qPCR |
| spellingShingle | Hui Zhou Xin Yang Jiayi Yu Jingyi Xu Ruiwen Zhang Ting Zhang Xijie Wang Jing Ma Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model Veterinary Medicine and Science circulating miRNA middle cerebral artery occlusion reference gene RT‐qPCR |
| title | Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model |
| title_full | Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model |
| title_fullStr | Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model |
| title_full_unstemmed | Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model |
| title_short | Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model |
| title_sort | reference gene identification for normalisation of rt qpcr analysis in plasma samples of the rat middle cerebral artery occlusion model |
| topic | circulating miRNA middle cerebral artery occlusion reference gene RT‐qPCR |
| url | https://doi.org/10.1002/vms3.879 |
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