Lopes Filipa M, Kimber Susan J, Bantounas Ioannis
Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, and the Manchester Academic Health Science Centre, Manchester, UK.
Bio Protoc. 2021 Sep 5;11(17):e4150. doi: 10.21769/BioProtoc.4150.
MicroRNAs are small RNAs that negatively regulate gene expression and play an important role in fine-tuning molecular pathways during development. There is increasing interest in studying their function in the kidney, but the majority of studies to date use kidney cell lines and assess the total amounts of miRNAs of interest either by qPCR or by high-throughput methods such as next generation sequencing. However, this provides little information as to the distribution of the miRNAs in the developing kidney, which is crucial in deciphering their role, especially as there are multiple kidney cell types, each with its own specific transcriptome. Thus, we present a protocol for obtaining spatial information for miRNA expression during kidney development by hybridization (ISH) of anti-miRNA, digoxigenin-labelled (DIG), Locked Nucleic Acid (LNA) probes on (i) native human embryonic tissue and (ii) human pluripotent stem cell (hPSC)-derived 3D kidney organoids that model kidney development. We found that the method reveals the precise localization of miRNA in specific anatomical structures and/or cell types and confirms their absence from others, thus informing as to their specific role during development.
微小RNA是一类小RNA，它们通过负向调控基因表达，在发育过程中对分子通路的精细调节发挥重要作用。目前，对于微小RNA在肾脏中功能的研究兴趣日益浓厚，但迄今为止，大多数研究使用肾细胞系，并通过定量聚合酶链反应（qPCR）或下一代测序等高通量方法来评估感兴趣的微小RNA的总量。然而，这对于微小RNA在发育中的肾脏中的分布情况提供的信息很少，而这对于解读它们的作用至关重要，特别是因为肾脏中有多种细胞类型，每种细胞类型都有其独特的转录组。因此，我们提出了一种方案，通过在（i）天然人类胚胎组织和（ii）模拟肾脏发育的人多能干细胞（hPSC）来源的三维肾脏类器官上，使用抗微小RNA、地高辛标记（DIG）的锁核酸（LNA）探针进行杂交（原位杂交，ISH），来获取肾脏发育过程中微小RNA表达的空间信息。我们发现，该方法揭示了微小RNA在特定解剖结构和/或细胞类型中的精确定位，并证实它们在其他结构或细胞类型中不存在，从而为它们在发育过程中的特定作用提供了信息。
