Importance of reference gene selection for articular cartilage mechanobiology studies

Al‐Sabah, Ayesha; Stadnik, Paulina; Gilbert, Sophie; Duance, Victor Colin; Blain, Emma Jane

doi:10.1016/j.joca.2015.11.007

Cited by 39 publications

(39 citation statements)

References 31 publications

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“…Real-time PCR was performed using a MxPro3000 QPCR system (Agilent Technologies, Stockport, UK) using ReadyMix™ Taq PCR reaction mix in conjunction with a Taqman® gene expression assay (Hpn: Bt03260765_m1; ThermoFisher Scientific (Loughborough, UK)). 18 S was used as a reference gene as previously described 46 .…”

Section: Methodsmentioning

confidence: 99%

The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

Wilkinson

Désilets

Lin

et al. 2017

Sci Rep

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Increasing evidence implicates serine proteinases in the proteolytic cascades leading to the pathological destruction of extracellular matrices such as cartilage in osteoarthritis (OA). We have previously demonstrated that the type II transmembrane serine proteinase (TTSP) matriptase acts as a novel initiator of cartilage destruction via the induction and activation of matrix metalloproteinases (MMPs). Hepsin is another TTSP expressed in OA cartilage such that we hypothesized this proteinase may also contribute to matrix turnover. Herein, we demonstrate that addition of hepsin to OA cartilage in explant culture induced significant collagen and aggrecan release and activated proMMP-1 and proMMP-3. Furthermore, hepsin directly cleaved the aggrecan core protein at a novel cleavage site within the interglobular domain. Hepsin expression correlated with synovitis as well as tumour necrosis factor α expression, and was induced in cartilage by a pro-inflammatory stimulus. However, a major difference compared to matriptase was that hepsin demonstrated markedly reduced capacity to activate proteinase-activated receptor-2. Overall, our data suggest that hepsin, like matriptase, induces potent destruction of the extracellular matrix whilst displaying distinct efficiencies for the cleavage of specific substrates.

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Section: Methodsmentioning

confidence: 99%

The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

Wilkinson

Désilets

Lin

et al. 2017

Sci Rep

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“…The wrong choice of reference gene for the normalization of RT-qPCR data may give rise to wildly inaccurate results [ 4 ]. For example, hypoxanthine phosphoribosyltransferase (HPRT) was demonstrated to be a suitable reference gene in placental tissues from intrahepatic cholestasis of pregnancy [ 5 ] and in bladder cancer cells [ 6 ], while it was found to be unsuitable for articular cartilage [ 7 ]. GAPDH and β-actin, which are the most commonly used reference genes in molecular biological studies, have been demonstrated to be useful reference genes in human prostate cancer [ 8 ], while they have been found to be unsuitable for normalizing mRNA levels in human asthmatic airways [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of reference genes for gene expression studies in mouse and N2a cell ischemic stroke models using quantitative real-time PCR

Kang

et al. 2018

BMC Neurosci

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BackgroundReal-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes. Reliable RT-qPCR results largely depend on normalization to suitable reference genes. Middle cerebral artery occlusion (MCAO) and oxygen–glucose deprivation/reoxygenation (OGD/R) are models that are commonly used to study ischemic stroke. However, the proper reference genes for RNA analysis in these two models have not yet been determined.ResultsIn this study, we evaluated the expression levels of six candidate housekeeping genes and selected the most suitable reference genes for RT-qPCR analyses of the cortices of MCAO mice and OGD/R-injured N2a cells. Four software programs, geNorm, NormFinder, BestKeeper and RefFinder, were used to validate the stabilities of the candidate reference genes. The results revealed that HPRT and 18S were the most stable reference genes in the cortices of MCAO mice and that β-actin and cyclophilin were the most stable reference genes in the OGD/R-injured N2a cells; in contrast, GAPDH and Sdha were the least stable genes in the cortices of MCAO mice and the OGD/R-injured N2a cells, respectively. Moreover, a combination of HPRT, 18S and cyclophilin was most suitable for normalization in analyses of the cortices of MCAO mice, and a combination of β-actin, cyclophilin, GAPDH, and 18S was most suitable for analyses of the OGD/R-injured N2a cells.ConclusionsThis study provides appropriate reference genes for further RT-qPCR analyses of in vivo and in vitro ischemic stroke and demonstrates the necessity of validating reference genes for RNA analyses under variable conditions.Electronic supplementary materialThe online version of this article (10.1186/s12868-018-0403-6) contains supplementary material, which is available to authorized users.

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“…Quantitative polymerase chain reaction (qPCR) was performed using SYBR green detection (Brilliant III Ultra‐Fast SYBR® QPCR mix, Agilent Technologies) using intron‐spanning primers for genes of interest (Suppl. Table S3), on a QPCR machine (MxPro3000, Agilent Technologies). All reactions were carried out at an annealing temperature of 60°C unless specified otherwise (Suppl.…”

Section: Methodsmentioning

confidence: 99%

“…Primers (MWG‐Biotech AG, Germany), were used at a final concentration of 200 nM and validated using cDNA standard curves with all primer efficiencies between 90 and 110% . Data were normalized to 18s and β‐actin which were identified from 8 reference genes using RefFinder (http://150.216.56.64/referencegene.php) as maintaining stable expression under these experimental conditions . Fold‐change in expression of genes of interest was calculated using the 2 −ΔΔCT method, after normalization to the reference genes and presented relative to the uninjured contra‐lateral limb.…”

Section: Methodsmentioning

confidence: 99%

Inflammatory and degenerative phases resulting from anterior cruciate rupture in a non‐invasive murine model of post‐traumatic osteoarthritis

Gilbert

Bonnet

Stadnik

et al. 2018

Journal Orthopaedic Research

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Joint injury is the predominant risk factor for post‐traumatic osteoarthritis development (PTOA). Several non‐invasive mouse models mimicking human PTOA investigate molecular mechanisms of disease development; none have characterized the inflammatory response to this acute traumatic injury. Our aim was to characterize the early inflammatory phase and later degenerative component in our in vivo non‐invasive murine model of PTOA induced by anterior cruciate ligament (ACL) rupture. Right knees of 12‐week‐old C57Bl6 mice were placed in flexion at a 30° offset position and subjected to a single compressive load (12N, 1.4 mm/s) to induce ACL rupture with no obvious damage to surrounding tissues. Tissue was harvested 4 h post‐injury and on days 3, 14, and 21; contralateral left knees served as controls. Histological, immunohistochemical, and gene analyzes were performed to evaluate inflammatory and degenerative changes. Immunohistochemistry revealed time‐dependent expression of mature (F4/80 positive) and inflammatory (CD11b positive) macrophage populations within the sub‐synovial infiltrate, developing osteophytes, and inflammation surrounding the ACL in response to injury. Up‐regulation of genes encoding acute pro‐inflammatory markers, inducible nitric oxide synthase, interleukin‐6 and interleukin‐17, and the matrix degrading enzymes, ADAMTS‐4 and MMP3 was detected in femoral cartilage, concomitant with extensive cartilage damage and bone remodelling over 21‐days post‐injury. Our non‐invasive model describes pathologically distinct phases of the disease, increasing our understanding of inflammatory episodes, the tissues/cells producing inflammatory mediators and the early molecular changes in the joint, thereby defining the early phenotype of PTOA. This knowledge will guide appropriate interventions to delay or arrest disease progression following joint injury. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:2118–2127, 2018.

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Importance of reference gene selection for articular cartilage mechanobiology studies

Cited by 39 publications

References 31 publications

The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

Evaluation of reference genes for gene expression studies in mouse and N2a cell ischemic stroke models using quantitative real-time PCR

Inflammatory and degenerative phases resulting from anterior cruciate rupture in a non‐invasive murine model of post‐traumatic osteoarthritis

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