Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow.
Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow.
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DOI：
10.1093/nar/gkw545
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发表时间：
2016-07-27
影响因子：
14.9
通讯作者：
Pfaffl MW
中科院分区：
文献类型：
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作者：
Buschmann D;Haberberger A;Kirchner B;Spornraft M;Riedmaier I;Schelling G;Pfaffl MW
小RNA-Seq已成为转录组学、基因表达谱分析和生物标志物发现的有力工具。从液体活检中对无细胞核酸，特别是microRNA（miRNA）进行测序，还为分子诊断提供了令人兴奋的可能性，并可能有助于建立疾病特异性生物标志物特征。然而，小型RNA-Seq工作流程的复杂性带来了挑战和偏见，研究人员需要意识到这些挑战和偏见，以便生成高质量的数据。需要严格的标准化和广泛的验证，以保证研究结果的可靠性、再现性和可比性。基于有缺陷的实验条件的假设可能是不一致的，甚至是误导性的。与成熟的MIQE qPCR实验指南相比，这项工作旨在建立实验设计和分析前样品处理、文库制备和测序反应标准化以及促进数据分析的指南。我们强调了小型RNA-Seq实验中的瓶颈，指出了严格的质量控制和验证的重要性，并为差异表达分析和生物标志物发现提供了引物。遵循我们的建议将鼓励更好的测序实践，增加实验透明度，并导致更可重复的小RNA-Seq结果。这将最终提高生物标志物签名的有效性，并允许可靠和稳健的临床预测。
Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will encourage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions.