National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk, Republic of Korea.
PLoS One. 2013 Apr 15;8(4):e60758. doi: 10.1371/journal.pone.0060758. Print 2013.
In the investigation of the expression levels of target genes, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most accurate and widely used method. However, a normalization step is a prerequisite to obtain accurate quantification results from RT-qPCR data. Therefore, many studies regarding the selection of reference genes have been carried out. Recently, these studies have involved large-scale gene analysis methods such as microarray and next generation sequencing. In our previous studies, we analyzed large amounts of transcriptome data from the cynomolgus monkey. Using a modification of this large-scale transcriptome sequencing dataset, we selected and compared 12 novel candidate reference genes (ARFGAP2, ARL1, BMI1, CASC3, DDX3X, MRFAP1, ORMDL1, RSL24D1, SAR1A, USP22, ZC3H11A, and ZRANB2) and 4 traditionally used reference genes (ACTB, GAPDH, RPS19, and YWHAZ) in 13 different whole-body tissues by the 3 well-known programs geNorm, NormFinder, and BestKeeper. Combined analysis by these 3 programs showed that ADP-ribosylation factor GTPase activating protein 2 (ARFGAP2), morf4 family associated protein 1 (MRFAP1), and ADP-ribosylation factor-like 1 (ARL1) are the most appropriate reference genes for accurate normalization. Interestingly, 4 traditionally used reference genes were the least stably expressed in this study. For this reason, selection of appropriate reference genes is vitally important, and large-scale analysis is a good method for finding new candidate reference genes. Our results could provide reliable reference gene lists for future studies on the expression of various target genes in the cynomolgus monkey.
在靶基因表达水平的研究中，逆转录实时定量聚合酶链反应（RT-qPCR）是最准确和广泛使用的方法。然而，为了从 RT-qPCR 数据中获得准确的定量结果，需要进行标准化步骤。因此，已经进行了许多关于参考基因选择的研究。最近，这些研究涉及了大规模基因分析方法，如微阵列和下一代测序。在我们之前的研究中，我们分析了大量来自食蟹猴的转录组数据。使用对这个大规模转录组测序数据集的修改，我们选择并比较了 12 个新的候选参考基因（ARFGAP2、ARL1、BMI1、CASC3、DDX3X、MRFAP1、ORMDL1、RSL24D1、SAR1A、USP22、ZC3H11A 和 ZRANB2）和 4 个传统的参考基因（ACTB、GAPDH、RPS19 和 YWHAZ）在 13 种不同的全身组织中的表达。通过 3 个知名程序 geNorm、NormFinder 和 BestKeeper 的综合分析表明，ADP-核糖基化因子 GTP 酶激活蛋白 2（ARFGAP2）、morf4 家族相关蛋白 1（MRFAP1）和 ADP-核糖基化因子样 1（ARL1）是准确归一化的最合适的参考基因。有趣的是，在这项研究中，4 个传统的参考基因的表达最不稳定。因此，选择合适的参考基因至关重要，大规模分析是寻找新的候选参考基因的好方法。我们的结果可为今后研究食蟹猴各种靶基因的表达提供可靠的参考基因列表。
