ArticleClinical and experimental dental research2022
Evidence-based selection of reference genes for RT-qPCR assays in periodontal research.
Article in Clinical and experimental dental research, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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2 citing papers in PubMed.
- The ATP-mediated cytokine release by macrophages is down-modulated by unconventional α9* nicotinic acetylcholine receptors.Frontiers in immunology · 2025Article
- Evidence-based selection of reference genes for RT-qPCR assays in periodontal research.Clinical and experimental dental research · 2022Article
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Abstract
objectiveTo underline the necessity of adequate reference genes for real-time quantitative polymerase chain reaction (RT-qPCR) and evaluate a novel tool for condition-specific reference gene selection.
backgroundRT-qPCR is a commonly used experimental technique that allows for highly sensitive analysis of gene transcription. Moreover, the use of internal reference genes as a means for relative quantification has rendered RT-qPCR a straightforward method for a variety of sciences, including dentistry. However, the expressional stability of internal reference genes must be evaluated for every assay in order to account for possible quantification bias. MATERIALS AND
methodsHerein, we used the software tool RefGenes to identify putatively stable reference genes with the help of microarray datasets and evaluated them. Additionally, we propose an evidence-based workflow for adequate normalization of thusly identified genes. Human gingival fibroblasts (HGF-hTert), human acute leukemia-derived monocytes (THP-1), and telomerase immortalized gingival keratinocytes (TIGKs) were subjected to set-ups simulating various glycemic conditions and lipopolysaccharide challenges. Five common housekeeping genes (HKGs) and five genes from RefGenes were selected as targets and RT-qPCR was performed subsequently. Then, normalization algorithms Bestkeeper, Normfinder, and geNorm were used for further analysis of the putative reference gene stability.
resultsRefGenes-derived targets exhibited the highest stability values in THP-1 and TIGK cell lines. Moreover, unacceptable standard variations were observed for some common HKG like β-actin. However, common HKG exhibited good stability values in HGF-hTert cells.
conclusionThe results indicate that microarray-based preselection of putative reference genes is a valuable refinement for RT-qPCR studies. Accordingly, the present study proposes a straightforward workflow for evidence-based preselection and validation of internal reference genes.
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