Housekeeping gene validation for RT-qPCR studies on synovial fibroblasts derived from healthy and osteoarthritic patients with focus on mechanical loading

English version

Autoři: Ute Nazet ^aff001; Agnes Schröder ^aff001; Susanne Grässel ^aff002; Dominique Muschter ^aff002; Peter Proff ^aff001; Christian Kirschneck ^aff001
Působiště autorů: Department of Orthodontics, University Medical Centre of Regensburg, Regensburg, Germany ^aff001; Department of Orthopaedics, University of Regensburg and Centre for Biomedical Technology, BioPark, Regensburg, Germany ^aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0225790

Souhrn

Selection of appropriate housekeeping genes is essential for the validity of data normalization in reverse transcription quantitative PCR (RT-qPCR). Synovial fibroblasts (SF) play a mediating role in the development and progression of osteoarthritis (OA) pathogenesis, but there is no information on reliable housekeeping genes available. Therefore the goal of this study was to identify a set of reliable housekeeping genes suitable for studies of mechanical loading on SF from healthy and OA patients. Nine genes were evaluated towards expression stability and ranked according their relative stability determined by four different mathematical procedures (geNorm, NormFinder, BestKeeper and comparative ΔC_q). We observed that RPLP0 (ribosomal protein, large, P0) and EEF1A1 (eukaryotic translation elongation factor 1 alpha 1) turned out to be the genes with the most stable expression in SF from non-OA or OA patients treated with or without mechanical loading. According to geNorm two genes are sufficient for normalization throughout. Expression of one tested target gene varied considerably, if normalized to different candidate housekeeping genes. Our study provides a tool for accurate and valid housekeeping gene selection in gene expression experiments on SF from healthy and OA patients with and without mechanical loading in consistent with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and additionally demonstrates the impact of proper housekeeping gene selection on the expression of the gene of interest.

Klíčová slova:

Gene expression – Algorithms – Cell cultures – Fibroblasts – Protein translation – Osteoarthritis – RNA synthesis – Elongation factors

Zdroje

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Housekeeping gene validation for RT-qPCR studies on synovial fibroblasts derived from healthy and osteoarthritic patients with focus on mechanical loading

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