Kirschneck, Christian and Batschkus, Sarah and Proff, Peter and Koestler, Josef and Spanier, Gerrit and Schroeder, Agnes (2017) Valid gene expression normalization by RT-qPCR in studies on hPDL fibroblasts with focus on orthodontic tooth movement and periodontitis. SCIENTIFIC REPORTS, 7: 14751. ISSN 2045-2322,
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Meaningful, reliable and valid mRNA expression analyses by real-time quantitative PCR (RT-qPCR) can only be achieved, if suitable reference genes are chosen for normalization and if appropriate RT-qPCR quality standards are met. Human periodontal ligament (hPDL) fibroblasts play a major mediating role in orthodontic tooth movement and periodontitis. Despite corresponding in-vitro gene expression studies being a focus of interest for many years, no information is available for hPDL fibroblasts on suitable reference genes, which are generally used in RT-qPCR experiments to normalize variability between samples. The aim of this study was to identify and validate suitable reference genes for normalization in untreated hPDL fibroblasts as well as experiments on orthodontic tooth movement or periodontitis (Aggregatibacter actinomycetemcomitans). We investigated the suitability of 13 candidate reference genes using four different algorithms (geNorm, NormFinder, comparative Delta C-q and BestKeeper) and ranked them according to their expression stability. Overall PPIB (peptidylprolyl isomerase A), TBP (TATA-box-binding protein) and RPL22 (ribosomal protein 22) were found to be most stably expressed with two genes in conjunction sufficient for reliable normalization. This study provides an accurate tool for quantitative gene expression analysis in hPDL fibroblasts according to the MIQE guidelines and shows that reference gene reliability is treatment-specific.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | REAL-TIME PCR; LIGAMENT CELLS; QUANTITATIVE PCR; MINIMUM INFORMATION; HOUSEKEEPING GENES; UP-REGULATION; IN-VITRO; HYPOXIA; RNA; IDENTIFICATION; |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Kieferorthopädie Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene Medicine > Lehrstuhl für Mund-, Kiefer- und Gesichtschirurgie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 14 Dec 2018 13:19 |
| Last Modified: | 25 Feb 2019 15:04 |
| URI: | https://pred.uni-regensburg.de/id/eprint/1860 |
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