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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.

ABSTRACT: 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 ?Cq). 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.

SUBMITTER: Nazet U

PROVIDER: S-EPMC6897414 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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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.

Nazet Ute U Schröder Agnes A Grässel Susanne S Muschter Dominique D Proff Peter P Kirschneck Christian C

PloS one 20191206 12

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 patien ...[more]

PMID: 31809510

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Project description:ObjectiveMechanisms responsible for osteoarthritic (OA) pain remain poorly understood, and current analgesic therapies are often insufficient. This study was undertaken to characterize and pharmacologically test the pain phenotype of a noninvasive mechanical joint loading model of OA, thus providing an alternative murine model for OA pain.MethodsThe right knees of 12-week-old male C57BL/6 mice were loaded at 9N or 11N (40 cycles, 3 times per week for 2 weeks). Behavioral measurements of limb disuse and mechanical and thermal hypersensitivity were acquired before mechanical joint loading and monitored for 6 weeks postloading. The severity of articular cartilage lesions was determined postmortem with the Osteoarthritis Research Society International scoring system. To assess efficacy of various treatments for pain, 9N-loaded mice were treated for 4 weeks with diclofenac (10 mg/kg), gabapentin (100 mg/kg), or anti-nerve growth factor (anti-NGF) (3 mg/kg).ResultsMechanical hypersensitivity and weight bearing worsened significantly in 9N-loaded mice (n = 8) and 11N-loaded mice (n = 8) 2 weeks postloading, compared to baseline values and nonloaded controls. Maximum OA scores of ipsilateral knees confirmed increased cartilage lesions in 9N-loaded mice (mean ± SEM 2.8 ± 0.2; P < 0.001) and 11N-loaded mice (5.3 ± 0.3; P < 0.001), compared to nonloaded controls (1.0 ± 0.0). Gabapentin and diclofenac restored pain behaviors to baseline values after 2 weeks of daily treatment, and gabapentin was more effective than diclofenac. A single injection of anti-NGF alleviated nociception 2 days after treatment and remained effective for 2 weeks, with a second dose inducing stronger and more prolonged analgesia.ConclusionOur findings show that mechanical joint loading induces OA lesions in mice and a robust pain phenotype that can be reversed using analgesics known to alleviate OA pain in patients. This establishes the use of mechanical joint loading as an alternative model for the study of OA pain.

Project description:Osteoarthritis (OA) is a multifactorial disease depending on molecular, genetic, and environmental factors like mechanical strain. Next to the cartilage and the subchondral bone, OA also affects the synovium, which is critically involved in the maintenance of joint homeostasis. As there is a correlation between the extracellular sodium content in the knee joint and OA, this study investigates the impact of sodium on OA-associated processes like inflammation and bone remodeling without and with mechanical loading in synovial fibroblasts. For that purpose, murine synovial fibroblasts from the knee joint were exposed to three different extracellular sodium chloride concentrations (-20 mM, ±0 mM and +50 mM NaCl) in the absence or presence of compressive or intermittent tensile strain. In addition to the intracellular Na+ content and gene expression of the osmoprotective transcription factor nuclear factor of activated T cells 5 (Nfat5), the gene and protein expression of inflammatory mediators (interleukin-6 (IL6), prostaglandin endoperoxide synthase-2 (Ptgs2)/prostaglandin E2 (PGE2)), and factors involved in bone metabolism (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG)) were analyzed by qPCR and ELISA. Mechanical strain already increased intracellular Na+ and Nfat5 gene expression at standard salt conditions to levels obtained by exposure to increased extracellular Na+ content. Both high salt and compressive strain resulted in elevated IL6 and PGE2 release. Intermittent tensile strain did not increase Il6 mRNA expression or IL6 protein secretion but triggered Ptgs2 expression and PGE2 production. Increased extracellular Na+ levels and compressive strain increased RANKL expression. In contrast, intermittent tension suppressed RANKL expression without this response being subject to modification by extracellular sodium availability. OPG expression was only induced by compressive strain. Changes in extracellular Na+ levels modified the inflammatory response and altered the expression of mediators involved in bone metabolism in cells exposed to mechanical strain. These findings indicate that Na+ balance and Nfat5 are important players in synovial fibroblast responses to mechanical stress. The integration of Na+ and Na+-dependent signaling will help to improve the understanding of the pathogenesis of osteoarthritis and could lead to the establishment of new therapeutic targets.

Project description:Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.

Dataset Information

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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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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