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

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-qPCRquality 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 ΔC 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.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 . In orthodontics mechanical forces applied to the teeth result in tensile and pressure zones within the periodontal ligament (PDL) 2 . PDL fibroblasts react to this mechanical strain with an increased synthesis of proinflammatory enzymes, cytokines and chemokines 2-4 , triggering osteoclastogenesis. Bacterial toxins from periodontal pathogens in periodontitis, such as the gram-negative Aggregatibacter actinomycetemcomitans (Agac), the key pathogen in aggressive periodontitis 5 , can in a similar way stimulate PDL fibroblasts, which are thus essential both for mediating orthodontic tooth movement and bacterial periodontitis.Real-time quantitative PCR (RT-qPCR) and DNA microarray analysis are the methods of choice to analyse transcription of cellular genes 6,7 . In contrast to microarray analysis, which allows expression profiling of a high number of genes, RT-qPCR enables a precise quantification of gene expression differences in physiological, pathological and various experimental states [8][9][10] . However, a reliable RT-qPCR setup is necessary to achieve valid results. To improve quality and reproducibility of RT-qPCR experiments, Bustin et al. published the MIQE guidelines 11 in 2009, detailing the minimum information for publication of quantitative real-time PCR experiments. A view in current literature shows that m...

Sodium‐chloride‐induced effects on the expression profile of human periodontal ligament fibroblasts with focus on simulated orthodontic tooth movement

“…Primary PDLFs were cultivated under standard cell‐culture conditions [37°C, 5% carbon dioxide (CO 2 ), 100% water (H 2 O)] in Dulbecco's modified Eagle's minimum essential medium (DMEM) high glucose (Sigma‐Aldrich, Munich, Germany), supplemented with 10% fetal calf serum (FCS) (PAN‐Biotech, Aidenbach, Germany), 1% L‐glutamine (GE Healthcare Europe, Penzberg, Germany), 100 μ M ascorbic acid (Sigma‐Aldrich), and 1% antibiotics/antimycotics (Sigma‐Aldrich). The PDLFs were retrieved from periodontal ligament tissue remnants scraped off the roots of teeth extracted in our facility for medical reasons (retained or displaced third molars) and were characterized according to their expression of PDLF‐specific marker genes by PCR and by their morphology, as previously reported . To generate a cell pool, we co‐cultivated cell lines from six individual patients (three male, three female, age range 17–27 yr).…”

“…Then, 2 ml of DMEM per well was added and, after a 24‐h preincubation phase, cells were incubated for another 48 h with addition of either 0 mM NaCl (control) or 40 mM NaCl (Sigma‐Aldrich), with or without compressive orthodontic force application of 2 g cm −2 . The latter was exerted by a sterile glass disc of defined size and weight according to a well‐established and published method (Fig. ).…”

Heat shock protein 70 dampens the inflammatory response of human PDL cells to mechanical loading in vitro

“…Experimental set‐up for hPDL cell experiments. Experimental conditions tested: physiological cell culture conditions (adherently growing hPDL cells at 70% confluence in full medium) vs simulated orthodontic compressive force (force of 2 g/cm 2 ) applied by a 17.1 g glass disk according to the method of Kanzaki and co‐workers…”

“…Confluent hPDL cells were subjected to compressive forces according to the protocol introduced by Kanzaki and co-workers. 5,30 Round glass plates were placed on top of the cells before the addition of another 1 mL DMEM containing 0.1% FBS. For illustration purposes, please refer to Figure 1.…”

“…Following mechanical loading, the expression of pro-inflammatory cytokines was quantified at the transcriptional level by means of realtime-PCR as described above using the following primer sequences according to 32 F I G U R E 1 Experimental set-up for hPDL cell experiments. Experimental conditions tested: physiological cell culture conditions (adherently growing hPDL cells at 70% confluence in full medium) vs simulated orthodontic compressive force (force of 2 g/cm 2 ) applied by a 17.1 g glass disk according to the method of Kanzaki and co-workers 5,30 Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer's instructions in order to quantify the protein expression of IL-6 and IL-8 (Qiagen, Hilden, Germany) in supernatants of hPDL cells that had been exposed to compressive cell stress and/or 25 μm of the HSP70 inhibitor VER15008 before.…”