Weil Patrick P, Jaszczyszyn Yan, Baroin-Tourancheau Anne, Postberg Jan, Amar Laurence
Department of Paediatrics, HELIOS Medical Centre Wuppertal, Centre for Clinical and Translational Research (CCTR), Witten/Herdecke University Hospital, Centre for Biomedical Education and Research (ZBAF), Witten, Germany.
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
Methods Mol Biol. 2017;1654:179-196. doi: 10.1007/978-1-4939-7231-9_12.
Small and long noncoding RNAs (ncRNAs) are key regulators of gene expression. Variations in ncRNA expression patterns can consequently affect the control of many cellular processes. Not just since 2006, when Andrew Z Fire and Craig C Mello were jointly awarded The Nobel Prize in Physiology or Medicine for their discovery of RNA interference, great efforts were undertaken to unleash the biomedical applicability of small noncoding RNAs, in particular microRNAs. With the technological evolution of massive parallel sequencing technologies over the last years, which now are available for an increasing number of scientists, there is a demand for comprehensible and efficient workflows reliable even for unique and valuable clinical specimens. Here we describe a highly reproducible low-cost protocol for analyses of miRNA expression patterns using tagged cDNA libraries and a multiplex sequencing strategy following an Illumina-like protocol. This protocol easily allows the identification of expression differences from samples of tissues of 1-2 mm and fluids of 50-200 μL. We further provide entry points into useful computational biology applications, whose target groups explicitly involve non-bioinformaticians.
小的和长的非编码RNA（ncRNA）是基因表达的关键调节因子。ncRNA表达模式的变化因此会影响许多细胞过程的调控。自2006年安德鲁·Z·法尔和克雷格·C·梅洛因发现RNA干扰而共同获得诺贝尔生理学或医学奖以来，人们就一直在努力挖掘小非编码RNA（特别是微小RNA）在生物医学方面的适用性。随着过去几年大规模平行测序技术的技术发展，现在越来越多的科学家能够使用这些技术，因此需要一种即使对于独特且珍贵的临床样本也可靠的、易于理解且高效的工作流程。在此，我们描述了一种高度可重复的低成本方案，该方案使用带标签的cDNA文库并遵循类似Illumina的方案采用多重测序策略来分析miRNA表达模式。该方案能够轻松地从1 - 2毫米的组织样本和50 - 200微升的液体样本中鉴定出表达差异。我们还提供了有用的计算生物学应用的切入点，其目标群体明确包括非生物信息学家。
