Validation of reference genes for the relative quantification of gene expression in human epicardial adipose tissue.
Validation of reference genes for the relative quantification of gene expression in human epicardial adipose tissue.
复制标题
DOI：
10.1371/journal.pone.0032265
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Richard D
中科院分区：
文献类型：
--
作者：
Chechi K;Gelinas Y;Mathieu P;Deshaies Y;Richard D
相对定量是评估基因表达的常用方法，然而其准确性和可靠性取决于最佳内源对照基因的选择，并且这种选择不能先验地进行。关于用于涉及人心外膜脂肪组织的研究的合适参考基因的可用信息有限。本研究的目的是评估和确定最佳的参考基因，用于相对定量的基因表达在人类心外膜脂肪库的瘦，超重和肥胖的主题。使用实时PCR分析来定量一些常用的参考基因，包括18S、ACTB、RPL 27、HPRT、CYCA、GAPDH、RPLPO、POLR 2A和B2 M。这些基因的表达稳定性使用Genstrom、Normfinder和Bestkeeper算法进行评估。此外，通过将受试者随机分为n = 2和n = 33两个队列，研究了样本量对验证过程的影响。 CYCA、GAPDH和RPL 27被鉴定为所有三种算法和两种样本量共有的最稳定的基因。它们作为参考基因对的使用可能有助于在涉及人心外膜脂肪组织的研究中增强相对定量的稳健性。
Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33. CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue.