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Evidence-based selection of reference genes for RT-qPCR assays in periodontal research
Daniel Diehl1,2, Anton Friedmann2, Hagen S Bachmann1
1Center for Biomedical Education and Research (ZBAF), Institute of Pharmacology and Toxicology, Faculty of Health, Witten/Herdecke University, Witten, Germany.
Objective:
To 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.
Background:
RT-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 Methods:
Herein, 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.
Results:
RefGenes-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.
Conclusion:
The 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.
