Verifying the stability of selected genes for normalization in Q PCR experiments of Spodoptera frugiperda cells during AcMNPV infection.
Verifying the stability of selected genes for normalization in Q PCR experiments of Spodoptera frugiperda cells during AcMNPV infection.
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DOI：
10.1371/journal.pone.0108516
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Thiem SM
中科院分区：
文献类型：
--
作者：
Salem TZ;Allam WR;Thiem SM
在病毒感染过程中，寻找具有稳定转录产物的基因作为定量实时聚合酶链式反应(qRT-PCR)的参考基因是一项具有挑战性的工作。苜蓿夜蛾核型多角体病毒(AcMNPV)能在全球范围内关闭Sf21细胞中宿主基因的转录，并改变其细胞骨架。在这项研究中，选择了7个宿主基因作为qRT-PCR基因表达实验的参考。在我们之前的微阵列研究中，其中两个基因，无蜕皮素(ECD)和肌球蛋白显示出稳定的RNA水平，这两个基因的RNA水平分别为6，12和24 HPI，ECD为48 HPI。肌动蛋白、微管蛋白、3-磷酸甘油醛脱氢酶(GAPDH)和28S核糖体(28S)是qRT-PCR常用的参考基因。选择核糖体蛋白L35(L35)基因来检测核糖体蛋白基因是否具有与28S和18S rRNA相似的稳定的RNA转录水平，并验证微阵列数据。除了先前已知具有稳定转录水平的28S外，qRT-PCR显示ECD转录水平在AcMNPV感染的整个过程中保持不变。随着感染的进展，细胞骨架基因如肌动蛋白、微管蛋白和肌球蛋白的转录本急剧下降。GAPDH和L35转录本也随着时间的推移而下降。这些结果表明，ECD是AcMNPV感染果蝇细胞过程中进行qRT-PCR实验的可靠参考基因。虽然28S可以作为这些实验的参考基因，但由于其含量丰富，不如ECD有用，这可能会使建立准确的数据分析基准值变得困难。
It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. Autographa californica nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of Spodoptera frugiperda cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.
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