Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida Genetics Institute, Interdisciplinary Program in Biomedical Sciences Neuroscience, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA.
Comp Biochem Physiol B Biochem Mol Biol. 2020 Dec;250:110490. doi: 10.1016/j.cbpb.2020.110490. Epub 2020 Aug 13.
Reductionist approaches in physiology and biochemistry are essential for understanding how animals cope and adapt to their environments. Transcriptomics is no longer restricted to a select few, and accessibility and affordability continue to facilitate its rapid growth as a science. More than 6000 publications (a conservative estimate) over the past decade quantify the response of the transcriptome to a wide breadth of questions in animal physiology. Transcriptomes have been quantified under conditions of hypoxia, climate change, salinity, drought, environmental pollution, and ultraviolet radiation among others; these studies have greatly improved understanding of the molecular machinery required for organismal adaptation. These "snapshots in time" however are never complete as the transcriptome is exquisitely sensitive to an individual's current physiologic state. Animal physiologists new to the field must recognize limitations of transcriptome technologies and consider experimental designs that strengthen physiologic interpretation. Current estimates suggest that a sample size of 6 or more are required for RNA-seq experiments in order to capture the majority of differentially expressed genes confidently. "Outside-the-box" approaches for statistical analyses of data derived from RNA-seq should be explored, as studies continue to point out that high false discoveries rates are pervasive with RNA-seq studies, reminiscent of the early days of microarrays. Incorporating biological variability, rather than reducing it (i.e. pooling strategies), into experimental designs is essential. Moreover, real-time PCR must not be viewed as a "validation step" to justify low samples sizes, but rather an orthogonal method to strengthen biological interpretation. The use of proper experimental controls in transcriptomics studies (i.e. spike-in controls and technical replication) are recommended and there is a pressing need for inter-laboratory tests (round robin experiments) to quantify repeatability and to identify sources of transcriptome variation within the context of animal physiology. Testing the reproducibility of transcriptome experiments in light of physiology in non-model organisms would be a significant contribution to the community. Single cell transcriptomics and multiplexing barcoding strategies such as decode-seq are poised to further advance the reductionist view of animal physiology; researchers are encouraged to consult literature herein and elsewhere for guidance on best practices and limitations of transcriptome technologies when studying the physiology of animals.
在生理学和生物化学中，还原论方法对于理解动物如何应对和适应环境至关重要。转录组学不再局限于少数人，其可及性和可负担性继续促进其作为一门科学的快速发展。在过去十年中，超过 6000 篇出版物（保守估计）定量描述了转录组对动物生理学广泛问题的反应。已经在缺氧、气候变化、盐度、干旱、环境污染和紫外线辐射等条件下对转录组进行了量化研究；这些研究极大地提高了对生物体适应所需分子机制的理解。然而，这些“时间快照”从来都不是完整的，因为转录组对个体当前的生理状态极其敏感。新涉足该领域的动物生理学家必须认识到转录组技术的局限性，并考虑加强生理解释的实验设计。目前的估计表明，为了有信心地捕获大多数差异表达基因，RNA-seq 实验需要 6 个或更多的样本。应该探索用于分析 RNA-seq 数据的“非常规”统计分析方法，因为研究继续指出，RNA-seq 研究中的高假发现率普遍存在，这让人想起微阵列的早期。将生物变异性纳入实验设计中而不是将其降低（即合并策略）至关重要。此外，实时 PCR 不应被视为“验证步骤”来证明样本量低，而是作为加强生物学解释的正交方法。建议在转录组学研究中使用适当的实验对照（即掺入对照和技术重复），并且迫切需要进行实验室间测试（轮次实验），以量化重复性并确定动物生理学背景下转录组变异的来源。根据非模型生物的生理学测试转录组实验的可重复性将是对该领域的重大贡献。单细胞转录组学和多路复用条形码策略（如 decode-seq）有望进一步推进动物生理学的还原论观点；研究人员被鼓励参考本文和其他地方的文献，了解在研究动物生理学时转录组技术的最佳实践和局限性。
