Valid gene expression normalization by RT-qPCR in studies on hPDL fibroblasts with focus on orthodontic tooth movement and periodontitis

Christian Kirschneck,Gerrit Spanier,Peter Proff,Sarah Batschkus,Josef Köstler,Agnes Schröder

doi:10.1038/s41598-017-15281-0

Christian Kirschneck, Gerrit Spanier + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-15281-0

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Abstract

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 ΔCq 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.

Highlights

Orthodontics and periodontology are specialties of dentistry tending to the treatment of misaligned teeth/jaws and bacterially induced inflammation of the periodontal tissues, respectively, with several interactive associations existing[1]
For four of the 13 to be investigated candidate reference genes (UBC, glucuronidase beta (GUSB), ACTB, tubulin beta class I (TUBB)), it was not possible to design a specific primer pair meeting all specified quality criteria[11,39,40,41], which is why they were exempted from further qPCR analysis (Table 1)
TBP is a TATA-box-binding protein, which is required for the initiation of transcription by RNA polymerase II44, and RPL22 is a ribosomal protein[45,46], which is involved in the control of morphogenesis by regulating Smad[2] mRNA splicing[47]

Summary

Introduction

Orthodontics and periodontology are specialties of dentistry tending to the treatment of misaligned teeth/jaws and bacterially induced inflammation of the periodontal tissues (periodontitis), respectively, with several interactive associations existing[1]. Aspects such as RNA integrity, qPCR-efficiency, primer specifity or secondary structure analyses of primers and amplicons, limiting their scientific validity and reliability[6,7,12] This is the case in the field of dentistry, with RT-qPCR studies on cells of teeth and the surrounding periodontal tissue continuously increasing, in orthodontics[12,13,14,15,16,17,18] and periodontology[19,20,21,22,23,24]. We investigated the conformity and reliability of these algorithms for bioinformatical analyses of reference gene stability

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