Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells.
Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells.
复制标题
骨头挑选：评估参考基因对于颅缝早闭和骨相关组织和细胞中基因表达的 RT-qPCR 定量的重要性
DOI：
10.1186/1756-0500-5-222
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发表时间：
2012-05-08
影响因子：
1.8
通讯作者：
Powell BC
中科院分区：
文献类型：
--
作者：
Yang X;Hatfield JT;Hinze SJ;Mu X;Anderson PJ;Powell BC
背景RT-qPCR是一种常用的基因表达定量工具，但其准确性取决于用于标准化的参考基因的选择和稳定性(稳态表达水平)/S。到目前为止，在骨骼领域，很少有研究来确定最稳定的参考基因，通常，RT-qPCR数据被归一化为未经验证的参考基因，最常见的是GAPDH、ACTB和18个S rRNA。结果使用geNorm和Normfinder程序，评估了小鼠和人类基因在三种不同实验条件下的稳定性：1)Crouzon综合征(颅缝融合)小鼠模型的疾病进展，2)从颅缝融合患者分离的颅缝细胞的增殖培养，以及3)小鼠骨髓基质细胞系的成骨。我们证明经典的参考基因并不总是最“稳定”的基因，基因的“稳定性”高度依赖于实验条件。选择稳定的基因，单独或组合，然后用来标准化骨钙素和碱性磷酸酶基因在颅缝融合期间在颅缝融合小鼠模型中的表达数据，并对策略进行比较。值得注意的是，碱性磷酸酶和骨钙素的表达趋势在归一化到最不稳定、最稳定或三个最稳定的基因时有显著差异。结论为了最大限度地减少评估基因表达水平的误差，强烈建议对参考小组进行分析，然后对几个稳定的基因进行标准化，而不是对单个基因进行标准化。特别是，我们得出结论，使用单一的、未经验证的“管家”基因，如GAPDHACTB和18 S rRNA，目前在骨骼领域的研究人员普遍使用，当变化在2倍或更少时，可能会产生可靠性有问题的数据，应该适当谨慎地解释这些数据。
BackgroundRT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability.ResultsUsing the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most ‘stable’genes and that gene ‘stability’is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes.ConclusionTo minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated “housekeeping” genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution.