Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Cell Rep Methods. 2021 Sep 27;1(5). doi: 10.1016/j.crmeth.2021.100061. Epub 2021 Aug 2.
Epigenetic modifications control the stability and translation of mRNA molecules. Here, we present a microscopy-based platform for quantifying modified RNA molecules and for relating the modification patterns to single-cell phenotypes. We directly capture mRNAs from cell lysates on oligo-dT-coated coverslips, then visually detect and sequence individual mA-immunolabled transcripts without amplification. Integration of a nanoscale device enabled us to isolate single cells on the platform, and thereby relate single-cell mA modification states to gene expression signatures and cell surface markers. Application of the platform to MUTZ3 leukemia cells revealed a marked reduction in cellular mA levels as CD34 leukemic progenitors differentiate to CD14 myeloid cells. We then coupled single-molecule mA detection with fluorescence hybridization (FISH) to relate mRNA and mA levels of individual genes to single-cell phenotypes. This single-cell multi-modal assay suite can empower investigations of RNA modifications in rare populations and single cells.
表观遗传修饰控制着 mRNA 分子的稳定性和翻译。在这里，我们提出了一种基于显微镜的平台，用于定量修饰的 RNA 分子，并将修饰模式与单细胞表型联系起来。我们直接从细胞裂解物中在 oligo-dT 涂层盖玻片上捕获 mRNA，然后在不进行扩增的情况下可视化检测和测序单个 mA 免疫标记的转录本。纳米级设备的集成使我们能够在平台上分离单个细胞，从而将单细胞 mA 修饰状态与基因表达特征和细胞表面标志物联系起来。该平台在 MUTZ3 白血病细胞中的应用表明，随着 CD34 白血病祖细胞分化为 CD14 髓样细胞，细胞内 mA 水平明显降低。然后，我们将单分子 mA 检测与荧光杂交 (FISH) 相结合，将单个基因的 mRNA 和 mA 水平与单细胞表型联系起来。这种单细胞多模态分析套件可以为研究稀有群体和单细胞中的 RNA 修饰提供支持。
