18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells.
18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells.
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18S rRNA是基于流感病毒感染细胞的实时PCR的可靠归一化基因。
DOI：
10.1186/1743-422x-9-230
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发表时间：
2012-10-08
期刊：
影响因子：
4.8
通讯作者：
Chang KC
中科院分区：
文献类型：
--
作者：
Kuchipudi SV;Tellabati M;Nelli RK;White GA;Perez BB;Sebastian S;Slomka MJ;Brookes SM;Brown IH;Dunham SP;Chang KC
定量反转录PCR （qRT-PCR）的一个必要条件是用一个内参基因对数据进行归一化，该内参基因无论如何处理（如病毒感染）都是不变的。几项研究发现，在不同的实验环境下，常用的管家基因，如β -肌动蛋白（ACTB）和甘油醛-3-磷酸脱氢酶（GAPDH）的表达存在差异。然而，ACTB和GAPDH仍然广泛应用于宿主基因对病毒感染的反应研究，包括流感病毒。到目前为止，还没有详细的研究对流感病毒感染中常用的管家基因的适用性进行比较。本研究评估了几种常用的内控基因[ACTB、GAPDH、18S核糖体RNA （18S rRNA）、ATP合成酶、H+转运、线粒体F1复合体、β多肽（ATP5B）和ATP合成酶、H+转运、线粒体Fo复合体、C1亚基（9亚基）（ATP5G1）]，以确定在感染一系列甲型流感病毒亚型的人、猪、鸡和鸭细胞中表达最稳定的基因。研究了5种甲型流感病毒亚型感染的人支气管上皮细胞（HBECs）、猪气管上皮细胞（PTECs）和鸡鸭原代肺源性细胞中常用管家基因的相对表达稳定性。利用NormFinder和BestKeeper软件程序对病毒和模拟感染细胞的qRT-PCR数据进行分析，发现18S rRNA是HBECs、PTECs和禽肺细胞中最稳定的基因。基于感染一系列流感病毒的细胞培养模型（HBECs、PTECs、鸡和鸭肺细胞）的数据，我们发现18S rRNA是最稳定的qRT-PCR数据规范化参考基因。本研究中评估的其他管家基因（包括ACTB和GPADH）的表达水平受到流感病毒感染的高度影响，因此不能作为RNA正常化的可靠参考基因。
One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.