Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR
Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR
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DOI：
10.1111/j.1744-7917.2008.00227.x
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发表时间：
2008-10-01
期刊：
影响因子：
4
通讯作者：
Zhu, Li
中科院分区：
文献类型：
--
作者：
Wang, Gen-Hong;Xia, Qing-You;Zhu, Li
mRNA水平的基因表达量化对于后基因组研究非常重要，因为基因表达水平是靶基因特殊生物学功能的反映。用于基因表达定量的方法，如芯片或定量实时聚合酶链反应（qRT-PCR），需要稳定表达的内参基因。因此，寻找合适的控制基因是基因定量的关键。本研究对家蚕变质过程中的内参基因进行了全基因组调查。根据外部对照归一化的全基因组寡核苷酸微阵列，选择12个基因作为推定的内参基因。然后，采用qRT-PCR进一步验证和选择潜在的内参候选基因。采用geNorm 3.4和NormFinder软件对结果进行分析，筛选出稳定基因。最后，综合考虑各方面因素，选择翻译起始因子4A、翻译起始因子3亚基4和翻译起始因子3亚基5（以sw22934、sw14876和sw13956为代表）作为在所研究的发育阶段的可靠内参，而细胞质肌动蛋白（sw22671）作为先前研究中常用的内参基因，在所研究的发育阶段变化很大。在未来的研究中，我们建议使用这三个稳定内参基因的几何平均值作为变质作用不同发育阶段数据归一化的精确归一化因子。
Gene expression quantification at mRNA level is very important for post-genomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction (qRT-PCR), require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT-PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 (represented by sw22934, sw14876, and sw13956) were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin (sw22671), the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.