Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage
Real-time fluorescence quantitative PCR (RT-qPCR) is widely used to detect gene expression levels, and selection of reference genes is crucial to the accuracy of RT-qPCR results. Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that using the panel of reference genes for RT...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2020-12-01
|
| Series: | Frontiers in Genetics |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2020.590124/full |
| _version_ | 1819058371273162752 |
|---|---|
| author | Liang Liu Hui Han Qingxian Li Ming Chen Siqi Zhou Hui Wang Hui Wang Liaobin Chen Liaobin Chen |
| author_facet | Liang Liu Hui Han Qingxian Li Ming Chen Siqi Zhou Hui Wang Hui Wang Liaobin Chen Liaobin Chen |
| author_sort | Liang Liu |
| collection | DOAJ |
| description | Real-time fluorescence quantitative PCR (RT-qPCR) is widely used to detect gene expression levels, and selection of reference genes is crucial to the accuracy of RT-qPCR results. Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that using the panel of reference genes for RT-qPCR is conducive to obtaining accurate experimental results. However, the selection of the panel of reference genes for RT-qPCR in rat developing cartilage has not been well documented. In this study, we selected eight reference genes commonly used in rat cartilage from literature (GAPDH, ACTB, 18S, GUSB, HPRT1, RPL4, RPL5, and SDHA) as candidates. Then, we screened out the optimal panel of reference genes in female and male rat cartilage of fetus (GD20), juvenile (PW6), and puberty (PW12) in physiology with stability analysis software of genes expression. Finally, we verified the reliability of the selected panel of reference genes with the rat model of intrauterine growth retardation (IUGR) induced by prenatal dexamethasone exposure (PDE). The results showed that the optimal panel of reference genes in cartilage at GD20, PW6, and PW12 in physiology was RPL4 + RPL5, which was consistent with the IUGR model, and there was no significant gender difference. Further, the results of standardizing the target genes showed that RPL4 + RPL5 performed smaller intragroup differences than other panels of reference genes or single reference genes. In conclusion, we found that the optimal panel of reference genes in female and male rat developing cartilage was RPL4 + RPL5, and there was no noticeable difference before and after birth. |
| first_indexed | 2024-12-21T13:54:08Z |
| format | Article |
| id | doaj.art-034f2a8cc9b049e9a5c6c49ad8e0f673 |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-12-21T13:54:08Z |
| publishDate | 2020-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-034f2a8cc9b049e9a5c6c49ad8e0f6732022-12-21T19:01:36ZengFrontiers Media S.A.Frontiers in Genetics1664-80212020-12-011110.3389/fgene.2020.590124590124Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat CartilageLiang Liu0Hui Han1Qingxian Li2Ming Chen3Siqi Zhou4Hui Wang5Hui Wang6Liaobin Chen7Liaobin Chen8Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, ChinaDepartment of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, ChinaDepartment of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, ChinaDepartment of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, ChinaDepartment of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, ChinaDepartment of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan, ChinaHubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan, ChinaDepartment of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, ChinaHubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan, ChinaReal-time fluorescence quantitative PCR (RT-qPCR) is widely used to detect gene expression levels, and selection of reference genes is crucial to the accuracy of RT-qPCR results. Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that using the panel of reference genes for RT-qPCR is conducive to obtaining accurate experimental results. However, the selection of the panel of reference genes for RT-qPCR in rat developing cartilage has not been well documented. In this study, we selected eight reference genes commonly used in rat cartilage from literature (GAPDH, ACTB, 18S, GUSB, HPRT1, RPL4, RPL5, and SDHA) as candidates. Then, we screened out the optimal panel of reference genes in female and male rat cartilage of fetus (GD20), juvenile (PW6), and puberty (PW12) in physiology with stability analysis software of genes expression. Finally, we verified the reliability of the selected panel of reference genes with the rat model of intrauterine growth retardation (IUGR) induced by prenatal dexamethasone exposure (PDE). The results showed that the optimal panel of reference genes in cartilage at GD20, PW6, and PW12 in physiology was RPL4 + RPL5, which was consistent with the IUGR model, and there was no significant gender difference. Further, the results of standardizing the target genes showed that RPL4 + RPL5 performed smaller intragroup differences than other panels of reference genes or single reference genes. In conclusion, we found that the optimal panel of reference genes in female and male rat developing cartilage was RPL4 + RPL5, and there was no noticeable difference before and after birth.https://www.frontiersin.org/articles/10.3389/fgene.2020.590124/fullintrauterine growth retardationreal-time quantitative polymerase chain reactioncartilage developmentprenatal dexamethasone exposurethe panel of reference genes |
| spellingShingle | Liang Liu Hui Han Qingxian Li Ming Chen Siqi Zhou Hui Wang Hui Wang Liaobin Chen Liaobin Chen Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage Frontiers in Genetics intrauterine growth retardation real-time quantitative polymerase chain reaction cartilage development prenatal dexamethasone exposure the panel of reference genes |
| title | Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage |
| title_full | Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage |
| title_fullStr | Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage |
| title_full_unstemmed | Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage |
| title_short | Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage |
| title_sort | selection and validation of the optimal panel of reference genes for rt qpcr analysis in the developing rat cartilage |
| topic | intrauterine growth retardation real-time quantitative polymerase chain reaction cartilage development prenatal dexamethasone exposure the panel of reference genes |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2020.590124/full |
| work_keys_str_mv | AT liangliu selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT huihan selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT qingxianli selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT mingchen selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT siqizhou selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT huiwang selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT huiwang selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT liaobinchen selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage AT liaobinchen selectionandvalidationoftheoptimalpanelofreferencegenesforrtqpcranalysisinthedevelopingratcartilage |