Hrvatin Siniša, Deng Francis, O'Donnell Charles W, Gifford David K, Melton Douglas A
The Harvard Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States of America.
The Harvard Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States of America; Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
PLoS One. 2014 Mar 3;9(3):e89459. doi: 10.1371/journal.pone.0089459. eCollection 2014.
Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.
转录谱分析是细胞生物学研究中的一项关键技术，但它受到试剂可用性的限制，这些试剂需能够唯一识别特定细胞类型并从中分离出高质量的RNA。我们报告了一种细胞内分选后RNA分析方法（MARIS），该方法可在细胞固定、细胞内免疫荧光染色和荧光激活细胞分选（FACS）后生成用于转录组分析的高质量RNA。因此，MARIS可简单地基于独特细胞内蛋白质的免疫荧光标记，从许多其他难以获取的细胞类型中分离出高质量RNA。作为原理验证，我们从分选的人胚胎干细胞衍生的胰岛素表达细胞以及成人人类β细胞中分离出RNA。MARIS是一项基础分子生物学技术，可应用于多个生物学学科。
