Epigenetics Programme, Babraham Institute, Cambridge, United Kingdom.
PLoS One. 2021 Jan 22;16(1):e0240769. doi: 10.1371/journal.pone.0240769. eCollection 2021.
A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal-a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.
从经过固定、染色和流式细胞分选的细胞中提取高质量 RNA 的简单方法将允许对高度纯化的细胞群和单个细胞进行常规转录组分析。然而，甲醛固定会损害 RNA 提取并抑制 RNA 扩增。在这里，我们表明，如果用乙二醛代替甲醛，一种广泛适用于免疫荧光染色方法的特征良好的固定剂，那么从染色和分选的哺乳动物细胞中可以很容易地提取到高质量的 RNA。尽管甲醛和乙二醛都能有效地形成蛋白质-蛋白质交联，但乙二醛不会将 RNA 与蛋白质交联，也不会形成稳定的 RNA 加合物，从而确保 RNA 在乙二醛固定后仍然可以被酶处理。我们发现，RNA 完整性通过乙二醛固定、甲醇或皂苷通透化、间接免疫荧光染色和流式分选得以维持。然后可以通过标准方法提取 RNA，并使用商业试剂盒将其加工成 RNA-seq 文库；通过 poly(A)+ RNA-seq 测量的 mRNA 丰度在新鲜收获的细胞和固定、染色和分选的细胞之间相关性良好。我们通过对 MCF-7 细胞进行细胞内 G2/M 特异性抗原细胞周期蛋白 B1（CCNB1）的染色来验证这种方法在流式细胞术上的适用性，并根据转录组数据显示 G2/M 期细胞的强烈富集。用 RNA 兼容的染色方法切换到乙二醛固定只需要对大多数现有的染色和分选方案进行微小的调整，这应该有助于对分选细胞进行常规转录组分析。
