Selection of appropriate reference genes in different tissues of Vaccinium dunalianum Wight by quantitative real-time PCR for gene expression studies
Selection of appropriate reference genes in different tissues of Vaccinium dunalianum Wight by quantitative real-time PCR for gene expression studies
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DOI：
10.1139/cjps-2020-0302
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发表时间：
2022-01
影响因子：
1.2
通讯作者：
Yalin Shao;Aiyi Chen;W. Chang;Guoze Li;Jinde Yu;Ping Zhao;Yong Ding
中科院分区：
文献类型：
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作者：
Yalin Shao;Aiyi Chen;W. Chang;Guoze Li;Jinde Yu;Ping Zhao;Yong Ding
摘要：实时定量聚合酶链反应（PCR）是研究基因表达相关变化的有力工具。目前，尚无关于dunalianum Wight （V. dunalianum Wight）内参基因的报道。本研究利用15个常用内参基因的表达谱，在云南和武定等地采集的褐花苜蓿不同组织中筛选合适的内参基因。比较分析不同样本中基因的循环阈值（Ct）。此外，使用BestKeeper、NormFinder和geNorm软件评估每个候选内参基因的稳定性。geNorm分析显示甘油醛-3-磷酸脱氢酶（GAPDH）和蛋白磷酸酶2A-2 （PP2A-2）的表达水平最稳定。通过NormFinder分析真核起始因子-2 （eukaryotic initiation factor-2, EIF-4A-2）的表达水平最为稳定，而Bestkeeper分析显示actin-related protein-1 （ARP-1）的表达水平最为稳定。综合分析和验证实验表明，EIF-4A-2和PP2A-2是dunalium Wight最合适的内参基因。
Abstract: Quantitative real-time polymerase chain reaction (PCR) is a powerful tool for investigating relevant changes in gene expression. At present, there are no reports on reference genes for Vaccinium dunalianum Wight (V. dunalianum Wight). Here, the expression profiles of 15 frequently used reference genes were used to screen for appropriate reference genes in different tissues of V. dunalianum Wight collected in Yunnan and Wuding, China. The cycle threshold (Ct) values of genes in different samples were compared and analyzed. Furthermore, BestKeeper, NormFinder, and geNorm software were used to evaluate the stability of each candidate reference gene. Analysis using geNorm showed that the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protein phosphatase 2A-2 (PP2A-2) were the most stable. In turn, the expression level of eukaryotic initiation factor-2 (EIF-4A-2) was the most stable as per analysis by NormFinder, while Bestkeeper revealed that actin-related protein-1 (ARP-1) was the most stably expressed. Comprehensive analysis and validation experiments showed that EIF-4A-2 and PP2A-2 were the most suitable reference genes for V. dunalianum Wight.