College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China.
Mycological Research Center, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China.
Sci Rep. 2016 Jul 12;6:29236. doi: 10.1038/srep29236.
The selection of appropriate internal control genes (ICGs) is a crucial step in the normalization of real-time quantitative PCR (RT-qPCR) data. Housekeeping genes are habitually selected for this purpose, despite accumulating evidence on their instability. We screened for novel, robust ICGs in the mushroom forming fungus Volvariella volvacea. Nine commonly used and five newly selected ICGs were evaluated for expression stability using RT-qPCR data in eight different stages of the life cycle of V. volvacea. Three different algorithms consistently determined that three novel ICGs (SPRYp, Ras and Vps26) exhibited the highest expression stability in V. volvacea. Subsequent analysis of ICGs in twenty-four expression profiles from nine filamentous fungi revealed that Ras was the most stable ICG amongst the Basidiomycetous samples, followed by SPRYp, Vps26 and ACTB. Vps26 was expressed most stably within the analyzed data of Ascomycetes, followed by HH3 and β-TUB. No ICG was universally stable for all fungal species, or for all experimental conditions within a species. Ultimately, the choice of an ICG will depend on a specific set of experiments. This study provides novel, robust ICGs for Basidiomycetes and Ascomycetes. Together with the presented guiding principles, this enables the efficient selection of suitable ICGs for RT-qPCR.
选择合适的内参基因（ICGs）是实时定量 PCR（RT-qPCR）数据归一化的关键步骤。尽管有越来越多的证据表明管家基因不稳定，但它们通常被用于此目的。我们筛选了蘑菇形成真菌草菇中的新型稳健 ICG。使用 RT-qPCR 数据评估了 8 个不同生活阶段的 V. volvacea 中的 9 个常用和 5 个新选择的 ICG 的表达稳定性。三种不同的算法一致确定，三个新型 ICG（SPRYp、Ras 和 Vps26）在 V. volvacea 中表现出最高的表达稳定性。随后对来自 9 种丝状真菌的 24 个表达谱中的 ICGs 进行分析，结果表明，Ras 是担子菌样品中最稳定的 ICG，其次是 SPRYp、Vps26 和 ACTB。在分析的子囊菌数据中，Vps26 的表达最稳定，其次是 HH3 和 β-TUB。没有一种 ICG 对所有真菌物种或同一物种的所有实验条件都普遍稳定。最终，ICG 的选择将取决于特定的实验集。本研究为担子菌和子囊菌提供了新型稳健的 ICG。结合提出的指导原则，这使得为 RT-qPCR 选择合适的 ICG 变得更加高效。
