A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.
A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.
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DOI：
10.1371/journal.pone.0160173
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Butter C
中科院分区：
文献类型：
--
作者：
Staines K;Batra A;Mwangi W;Maier HJ;Van Borm S;Young JR;Fife M;Butter C
实时定量 PCR 检测广泛用于禽类实验样品中 mRNA 的定量。为代表性样本中变异最低而选择的多个稳定表达的参考基因可用于控制随机技术变异。参考基因检测必须可靠、具有高扩增特异性和效率，并且不会产生来自污染 DNA 的信号。虽然最近的研究论文识别了在特定组织和实验治疗中稳定的特定基因，但在这里我们描述了一组十个鸟类基因引物和探针组，可用于在许多实验环境中识别合适的参考基因。该面板在两个实验场景中使用 TaqMan 和 SYBR Green 系统进行了测试：组织收集和培养的成纤维细胞的病毒感染。 GeNorm 和 NormFinder 算法能够在每种情况下选择适当的参考基因集。我们展示了使用所选基因对检测表达的统计显着差异的影响。将结果与使用 28s 核糖体 RNA（目前养鸡工作中最广泛接受的参考基因）获得的结果进行比较，确定其使用可能会产生误导性结果的情况。消除 RNA 的 DNA 污染的方法减少了可检测的 DNA，但并未完全去除。因此，我们特别重视使用 DNA 模板测试每个 qPCR 测定是否存在信号。这里开发的测定和分析为选择鸟类生物学研究的参考基因提供了有用的资源。
Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology.