Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus
Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus
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DOI：
10.1016/j.vetmic.2005.06.014
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发表时间：
2005-10-31
影响因子：
3.3
通讯作者：
Zhang, MF
中科院分区：
文献类型：
--
作者：
Li, YP;Bang, DD;Zhang, MF
传染性法氏囊病病毒（IBDV）可引起鸡的疾病，其特点是免疫抑制和高死亡率。目前，实时RT-PCR已被用于定量病毒特异性RNA和更好地了解宿主对感染的反应。然而，定量实时RT-PCR的规范化需要一个合适的内部控制。因此，我们研究了感染IBDV 7天后鸡胚（CE）细胞培养中p -肌动蛋白、28S rRNA、18S rRNA、甘油醛-3-磷酸脱氢酶（GAPDH）、TATA盒结合蛋白（TBP）和β -2微球蛋白等6种鸡基因的表达模式。将不同感染倍数（MOI）的IBDV疫苗株Bursine-2接种于CE细胞，以归一化（100 ng）和非归一化（10 μ l）总RNA合成cDNA，采用实时荧光定量pcr检测基因表达。结果表明，β -肌动蛋白、28S rRNA、18S rRNA和GAPDH是持续表达最多的基因，而TBP和β -2微球蛋白在感染过程中被显著诱导。在这些恒定表达的基因中，28S rRNA和18S rRNA高表达；β -肌动蛋白中等表达，GAPDH在CE细胞培养中表达水平较低。此外，β -肌动蛋白在归一化和非归一化试验中均无显著变化，且与病毒剂量无关，而其他基因则有。在CE细胞培养中，β -肌动蛋白被成功地用作定量法氏囊素-2病毒特异性RNA载量的内对照。因此，β -肌动蛋白被认为是研究IBDV感染后CE细胞基因表达和病毒特异性RNA载量的合适内对照。(c) 2005 Elsevier B.V.版权所有
Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real-time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including P-actin, 28S rRNA, 18S rRNA, glyceral dehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 mu l) total RNA. The results showed that beta-actin, 28S rRNA, 18S rRNA and GAPDH were the most constantly expressed genes, while TBP and beta-2-microglobulin were markedly induced during the infection course. Of these constant expressed genes, 28S rRNA and 18S rRNA are highly expressed; beta-actin intermediately expressed and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2 virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection. (c) 2005 Elsevier B.V. All rights reserved.