Gapdh Gene Expression Is Modulated by Inflammatory Arthritis and Is not Suitable for qPCR Normalization

Montero‐Melendez, Trinidad; Perretti, Mauro

doi:10.1007/s10753-014-9829-x

Cited by 20 publications

(16 citation statements)

References 45 publications

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“…Furthermore, HPRT and PPIA have previously been identified as candidate reference genes in the ATDC5 chondroprogenitor cell line 21 . In an in vivo model of inflammatory joint pathology (K/BxN), GAPDH transcript levels were significantly reduced whereas HPRT, along with β 2 -microglobulin and RPL13a were validated as most constant in expression 29 , again highlighting how the model system impacts on the selection of reference gene(s) used.…”

Section: Discussionmentioning

confidence: 99%

Importance of reference gene selection for articular cartilage mechanobiology studies

Al‐Sabah

Stadnik

Gilbert

et al. 2016

Osteoarthritis and Cartilage

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SummaryObjectiveIdentification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively.DesignThe expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder.ResultsAppropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions.ConclusionThis study demonstrates that commonly used ‘reference genes’ may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation.

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Section: Discussionmentioning

confidence: 99%

Importance of reference gene selection for articular cartilage mechanobiology studies

Al‐Sabah

Stadnik

Gilbert

et al. 2016

Osteoarthritis and Cartilage

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“…GAPDH was found to be unsuitable for normalization in skeletal muscle development in pig [ 48 ]; similar results were reported in goat [ 16 ] and cattle [ 51 ]. In addition, GAPDH was observed to be up-regulated and down-regulated under various conditions [ 7 , 9 , 52 , 53 ]. Likewise, a remarkable change in the expression of 18S occurred during osteoblast differentiation, with a continual increase in expression observed over time [ 6 ].…”

Section: Discussionmentioning

confidence: 99%

Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

et al. 2015

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The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

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“…Real time-PCR was performed with Power SYBR Green PCR MasterMix (Applied Biosystems) on the ABI Prism 7900HT Sequence Detection System. Expression was calculated as 2 −ΔΔCt 53 using HPRT as a reference gene 54 . Quantitect primers (QIAGEN) used are shown in Table S2, with an annealing temperature of 55°C used for all primers.…”

Section: Methodsmentioning

confidence: 99%

Therapeutic senescence via GPCR activation in synovial fibroblasts facilitates resolution of arthritis

et al. 2020

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Rheumatoid arthritis affects individuals commonly during the most productive years of adulthood. Poor response rates and high costs associated with treatment mandate the search for new therapies. Here we show that targeting a specific G-protein coupled receptor promotes senescence in synovial fibroblasts, enabling amelioration of joint inflammation. Following activation of the melanocortin type 1 receptor (MC 1), synovial fibroblasts acquire a senescence phenotype characterized by arrested proliferation, metabolic reprogramming and marked gene alteration resembling the remodeling phase of wound healing, with increased matrix metalloproteinase expression and reduced collagen production. This biological response is attained by selective agonism of MC 1 , not shared by non-selective ligands, and dependent on downstream ERK1/2 phosphorylation. In vivo, activation of MC 1 leads to anti-arthritic effects associated with induction of senescence in the synovial tissue and cartilage protection. Altogether, selective activation of MC 1 is a viable strategy to induce cellular senescence, affording a distinct way to control joint inflammation and arthritis.

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Gapdh Gene Expression Is Modulated by Inflammatory Arthritis and Is not Suitable for qPCR Normalization

Cited by 20 publications

References 45 publications

Importance of reference gene selection for articular cartilage mechanobiology studies

Importance of reference gene selection for articular cartilage mechanobiology studies

Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

Therapeutic senescence via GPCR activation in synovial fibroblasts facilitates resolution of arthritis

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