Importance of reference gene selection for articular cartilage mechanobiology studies.
Importance of reference gene selection for articular cartilage mechanobiology studies.
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DOI：
10.1016/j.joca.2015.11.007
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发表时间：
2016-04
影响因子：
7
通讯作者：
Blain EJ
中科院分区：
文献类型：
--
作者：
Al-Sabah A;Stadnik P;Gilbert SJ;Duance VC;Blain EJ
在关节软骨的力学-生物学途径中差异表达的基因的鉴定提供了对骨关节炎（OA）开始和/或进展背后的分子机制的深入了解。定量PCR（qPCR）通常用于测量基因表达，并且依赖于使用参考基因进行标准化。参考基因稳定性的适当验证对于准确的数据分析和解释至关重要。本研究分别测定了关节软骨外植体和原代软骨细胞在不同压缩载荷和拉伸应变下的体外参考基因稳定性。通过qPCR测定八种常用参考基因（18 s、ACTB、GAPDH、HPRT 1、PPIA、RPL 4、SDHA和YWHAZ）的表达，并使用四种软件包（比较delta-Ct方法、geNorm、Normalization和BestKeeper）比较数据。使用Referral计算排序权重的几何平均值。用于关节软骨组织或分离的软骨细胞中机械调节转录物水平正常化的适当参考基因取决于实验设置。SDHA、YWHAZ和RPL 4是最稳定的基因，而甘油醛-3-磷酸脱氢酶（GAPDH）和次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）在较小程度上显示响应于负荷的可变表达，证明它们在这种体外研究中的不适合性。使用不稳定的参考基因使聚集蛋白聚糖（ACAN）和基质金属蛋白酶3（MMP 3）的表达正常化的效果导致这些机械敏感基因的不准确定量和错误的解释/结论。这项研究表明，常用的“参考基因”可能不适合体外软骨软骨细胞机械生物学研究，加强了以下原则，即在每次实验之前仔细验证参考基因是必不可少的，以获得稳健和可重复的qPCR数据进行分析/解释。
Identification 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. The 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. Appropriate 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. This 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.