miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage
miRNAs are emerging as key regulators of complex biological systems in several developmental processes. qRT-PCR is a powerful tool to quantitatively assess the profiles and modulation of miRNA expression. In the emerging field of cartilage maturation studies, from precursor to hypertrophic chondrocy...
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MDPI AG
2019-06-01
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| Online Access: | https://www.mdpi.com/2073-4409/8/6/631 |
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| author | Enrico Ragni Paola De Luca Antongiulio Marmotti Laura de Girolamo |
| author_facet | Enrico Ragni Paola De Luca Antongiulio Marmotti Laura de Girolamo |
| author_sort | Enrico Ragni |
| collection | DOAJ |
| description | miRNAs are emerging as key regulators of complex biological systems in several developmental processes. qRT-PCR is a powerful tool to quantitatively assess the profiles and modulation of miRNA expression. In the emerging field of cartilage maturation studies, from precursor to hypertrophic chondrocytes, few data about miRNA regulation are available, and no consensus on the best reference gene (RG) has been reached. This is a crucial pitfall since reliable outcomes depend on proper data normalization. The aim of this work was to identify reliable and stable miRNA RGs, basing the analysis on available high throughput qRT-PCR miRNA data (from the NCBI Gene Expression Omnibus database, GSE49152) obtained from human embryonic cartilage tissues enriched in the precursor, differentiated, and hypertrophic chondrocytes. Four normalization approaches were used, and the stability was quantified by combining BestKeeper, delta-Ct, geNorm, and NormFinder statistical tools. An integrated approach allowed to identify miR-26a-5p as the most stable RG and miR-212-3p as the worst one. RNU44, used in original dataset analysis, performed as second best RG. Applications of different normalization strategies significantly impacted the profiles and modulation of miRNA expression. Herein presented results point out the crucial need of a consensus on data normalization studies aimed at dissecting miRNA role in human cartilage development, to avoid the postulation of unreliable biological conclusions. |
| first_indexed | 2024-03-12T09:08:28Z |
| format | Article |
| id | doaj.art-f066b95c60a9434aa96181b159212a92 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-12T09:08:28Z |
| publishDate | 2019-06-01 |
| publisher | MDPI AG |
| record_format | Article |
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| spelling | doaj.art-f066b95c60a9434aa96181b159212a922023-09-02T15:10:08ZengMDPI AGCells2073-44092019-06-018663110.3390/cells8060631cells8060631miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human CartilageEnrico Ragni0Paola De Luca1Antongiulio Marmotti2Laura de Girolamo3IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, I-20161 Milano, ItalyIRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, I-20161 Milano, ItalyDepartment of Orthopaedics and Traumatology, University of Turin, I-10124 Torino, ItalyIRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, I-20161 Milano, ItalymiRNAs are emerging as key regulators of complex biological systems in several developmental processes. qRT-PCR is a powerful tool to quantitatively assess the profiles and modulation of miRNA expression. In the emerging field of cartilage maturation studies, from precursor to hypertrophic chondrocytes, few data about miRNA regulation are available, and no consensus on the best reference gene (RG) has been reached. This is a crucial pitfall since reliable outcomes depend on proper data normalization. The aim of this work was to identify reliable and stable miRNA RGs, basing the analysis on available high throughput qRT-PCR miRNA data (from the NCBI Gene Expression Omnibus database, GSE49152) obtained from human embryonic cartilage tissues enriched in the precursor, differentiated, and hypertrophic chondrocytes. Four normalization approaches were used, and the stability was quantified by combining BestKeeper, delta-Ct, geNorm, and NormFinder statistical tools. An integrated approach allowed to identify miR-26a-5p as the most stable RG and miR-212-3p as the worst one. RNU44, used in original dataset analysis, performed as second best RG. Applications of different normalization strategies significantly impacted the profiles and modulation of miRNA expression. Herein presented results point out the crucial need of a consensus on data normalization studies aimed at dissecting miRNA role in human cartilage development, to avoid the postulation of unreliable biological conclusions.https://www.mdpi.com/2073-4409/8/6/631miRNAcartilagereference geneqRT-PCRdevelopmentchondrocyte |
| spellingShingle | Enrico Ragni Paola De Luca Antongiulio Marmotti Laura de Girolamo miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage Cells miRNA cartilage reference gene qRT-PCR development chondrocyte |
| title | miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage |
| title_full | miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage |
| title_fullStr | miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage |
| title_full_unstemmed | miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage |
| title_short | miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage |
| title_sort | mir 26a 5p is a stable reference gene for mirna studies in chondrocytes from developing human cartilage |
| topic | miRNA cartilage reference gene qRT-PCR development chondrocyte |
| url | https://www.mdpi.com/2073-4409/8/6/631 |
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