Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages
Abstract Background Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental st...
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| Format: | Article |
| Language: | English |
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BMC
2022-08-01
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| Series: | BMC Genomics |
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| Online Access: | https://doi.org/10.1186/s12864-022-08830-z |
| _version_ | 1798042496556597248 |
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| author | Jeongah Song Jeonghee Cho Jeongsik Park Jeong Ho Hwang |
| author_facet | Jeongah Song Jeonghee Cho Jeongsik Park Jeong Ho Hwang |
| author_sort | Jeongah Song |
| collection | DOAJ |
| description | Abstract Background Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. Results As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, β-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. Conclusions We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions. |
| first_indexed | 2024-04-11T22:36:19Z |
| format | Article |
| id | doaj.art-f69cc27ab0414f6eaf06bba1a596073d |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-11T22:36:19Z |
| publishDate | 2022-08-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-f69cc27ab0414f6eaf06bba1a596073d2022-12-22T03:59:13ZengBMCBMC Genomics1471-21642022-08-0123111310.1186/s12864-022-08830-zIdentification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stagesJeongah Song0Jeonghee Cho1Jeongsik Park2Jeong Ho Hwang3Animal Model Research Group, Korea Institute of ToxicologyAnimal Model Research Group, Korea Institute of ToxicologyAnimal Model Research Group, Korea Institute of ToxicologyAnimal Model Research Group, Korea Institute of ToxicologyAbstract Background Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. Results As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, β-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. Conclusions We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions.https://doi.org/10.1186/s12864-022-08830-zQuantitative real time PCRReference genesMinipigRefFinder |
| spellingShingle | Jeongah Song Jeonghee Cho Jeongsik Park Jeong Ho Hwang Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages BMC Genomics Quantitative real time PCR Reference genes Minipig RefFinder |
| title | Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages |
| title_full | Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages |
| title_fullStr | Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages |
| title_full_unstemmed | Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages |
| title_short | Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages |
| title_sort | identification and validation of stable reference genes for quantitative real time pcr in different minipig tissues at developmental stages |
| topic | Quantitative real time PCR Reference genes Minipig RefFinder |
| url | https://doi.org/10.1186/s12864-022-08830-z |
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