Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia.
College of Health and Biomedicine, Victoria University, Melbourne, Victoria, Australia.
PLoS One. 2018 May 10;13(5):e0196438. doi: 10.1371/journal.pone.0196438. eCollection 2018.
Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content.
在运动研究中，通过定量 PCR 对骨骼肌中的基因表达进行分析是常规操作。数据的可重复性和可靠性从根本上取决于实验的执行和解释方式。尽管该检测方法已经很普及，但不同实验室之间的实验方案和数据分析存在相当大的差异，而且整个检测过程缺乏一致性的适当质量控制步骤。在这项研究中，我们展示了定量 PCR 工作流程各个步骤的多项实验，并演示了如何在运动研究中使用人类骨骼肌样本进行定量 PCR 实验。我们还测试了在进行 qPCR 时的一些常见错误。有趣的是，我们发现，在提取 RNA 之前，将肌肉短时间（10 分钟）处理并不会影响 RNA 质量，并且分离出的总 RNA 在室温下可保存长达一周。我们的数据表明，使用不稳定的参考基因会导致最终结果出现显著差异。或者，可以使用 cDNA 含量进行数据归一化；然而，为了获得准确的 cDNA 含量，必须从 cDNA 样品中完全去除 RNA。
