Sylwestrak Emily Lauren, Rajasethupathy Priyamvada, Wright Matthew Arnot, Jaffe Anna, Deisseroth Karl
Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA.
Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA; Department of Psychiatry and Behavioral Sciences, Stanford University, 318 Campus Drive, Stanford, CA 94305, USA.
Cell. 2016 Feb 11;164(4):792-804. doi: 10.1016/j.cell.2016.01.038.
In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization over large volumes has been largely restricted to labeling of proteins. But volumetric nucleic acid labeling may represent a far greater scientific and clinical opportunity, enabling detection of not only diverse coding RNA variants but also non-coding RNAs. Moreover, scaling immunohistochemical detection to large tissue volumes has limitations due to high cost, limited renewability/availability, and restricted multiplexing capability of antibody labels. With the goal of versatile, high-content, and scalable molecular phenotyping of intact tissues, we developed a method using carbodiimide-based chemistry to stably retain RNAs in clarified tissue, coupled with amplification tools for multiplexed detection. The resulting technology enables robust measurement of activity-dependent transcriptional signatures, cell-identity markers, and diverse non-coding RNAs in rodent and human tissue volumes. The growing set of validated probes is deposited in an online resource for nucleating related developments from across the scientific community.
在最近开发的用于完整组织内高分辨率成像的方法中,对大体积组织进行分子表征在很大程度上仅限于蛋白质标记。但是,体积核酸标记可能代表着更大的科学和临床机遇,不仅能够检测多种编码RNA变体,还能检测非编码RNA。此外,将免疫组织化学检测扩展到大型组织体积存在局限性,原因在于成本高、可更新性/可用性有限以及抗体标记的多重检测能力受限。为了实现对完整组织进行通用、高内涵和可扩展的分子表型分析,我们开发了一种方法,利用基于碳二亚胺的化学方法在澄清的组织中稳定保留RNA,并结合用于多重检测的扩增工具。由此产生的技术能够在啮齿动物和人类组织体积中对活性依赖的转录特征、细胞身份标记物以及多种非编码RNA进行可靠测量。越来越多经过验证的探针被存入一个在线资源库,以推动整个科学界相关研究的开展。