Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.
Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.
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DOI：
10.1371/journal.pone.0112706
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Biffinger JC
中科院分区：
文献类型：
--
作者：
Cusick KD;Fitzgerald LA;Pirlo RK;Cockrell AL;Petersen ER;Biffinger JC
粗糙脉孢菌一直是研究生物节律途径的模式生物，最近由于其提高了纤维素酶的产生能力而在生物燃料行业获得了关注。然而，为了优化粗毛拟青霉的生物技术应用，必须解决不同环境条件下生长过程中的代谢途径。逆转录定量聚合酶链式反应(RT-qPCR)是一种提供高通量平台的技术，可以在此平台上测量一大组基因随时间的表达。选择合适的参考基因对于使用相对定量进行基因表达研究是至关重要的，因为这一策略是基于将目标基因的表达标准化为在实验条件下表达稳定的参考基因。这项研究评估了12个候选参考基因，当在不同的光和温度条件下，在连续培养的生物反应器中生长时，可以用于粗毛藻。根据NormFinder和Best Keeper软件包的综合稳定值，以下是在以下条件下最合适的参考基因：(1)光/暗循环：BTL、AS1和VMA1；(2)全暗生长：BTL、TBP、VMA1和VMA2；(3)温度通量：BTL、VMA1、ACT和ASL；(4)所有条件组合：VMA1、VMA2、TBP和BTL。由于粗糙奈瑟氏菌以不同的细胞类型(单核或多核)存在，因此使用绝对定量方法进一步评估了候选基因子集的表达变化。比值与阈值周期(CT值)之间存在很强的负相关，表明CT的变化是转录的可靠反映，而不是基因拷贝数的波动。这项研究的结果确定了适合作为参考基因的基因，用于粗劣新月球藻的RT-qPCR研究，并表明即使存在不同类型的细胞，相对定量也是一种可以接受的方法来测量生物反应器中生长过程中基因表达的变化。
Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene expression changes during growth in bioreactors.
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影响因子：
11.3
作者：
Baker CL;Loros JJ;Dunlap JC
通讯作者：
Dunlap JC
影响因子：
2.2
作者：
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DOI：
10.1073/pnas.1200785109
发表时间：
2012-05-08
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
2.7
作者：
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