Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry.
Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry.
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乙二醛固定有利于在抗原染色和流式细胞术细胞分选后进行转录组分析。
DOI：
10.1371/journal.pone.0240769
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Houseley J
中科院分区：
文献类型：
--
作者：
Channathodiyil P;Houseley J
从已经通过流式细胞术固定、染色和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细胞进行常规转录组分析。然而，甲醛固定损害RNA提取并抑制RNA扩增。在这里，我们表明，可以很容易地从染色和分选的哺乳动物细胞中提取高质量的RNA，如果甲醛被取代的glyphosate-一个良好的特征固定剂，是广泛兼容的免疫荧光染色方法。虽然甲醛和乙二醛都能有效地形成蛋白质-蛋白质交联，但乙二醛不会使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接近并易于酶促操作。我们发现RNA的完整性是通过乙二醛固定，甲醇或皂苷透化，间接免疫荧光染色和流式分选。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与固定、染色和分选的细胞之间具有良好的相关性。我们通过对MCF-7细胞内G2/M期特异性抗原细胞周期蛋白B1（CCNB 1）进行染色来验证这种方法对流式细胞术的适用性，并基于转录组学数据显示出对G2/M期细胞的强烈富集。切换到使用RNA兼容染色方法的乙二醛固定仅需要对大多数现有染色和分选方案进行微小调整，并且应该有助于对分选细胞进行常规转录组学分析。
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.
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影响因子：
5.4
作者：
Alles J;Karaiskos N;Praktiknjo SD;Grosswendt S;Wahle P;Ruffault PL;Ayoub S;Schreyer L;Boltengagen A;Birchmeier C;Zinzen R;Kocks C;Rajewsky N
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作者：
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作者：
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通讯作者：
Burnett, WV
影响因子：
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作者：
Ly, Tony;Whigham, Arlene;Lamond, Angus I.
通讯作者：
Lamond, Angus I.