Expression microdissection isolation of enriched cell populations from archival brain tissue.
Expression microdissection isolation of enriched cell populations from archival brain tissue.
复制标题
从档案脑组织中表达显微解剖分离富集的细胞群。
DOI：
10.1016/j.jneumeth.2016.05.007
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发表时间：
2016
影响因子：
3
通讯作者：
Appleby-Mallinder C
中科院分区：
文献类型：
--
作者：
Appleby-Mallinder C
背景激光捕获显微切割（LCM）是一种用于获取富集细胞群的成熟技术，可以进行进一步的下游分析，尽管它确实有局限性。表达显微切割 (xMD) 是一种新技术，开始解决这些缺陷，例如操作员依赖性和污染。新方法 xMD 利用免疫组织化学与显色剂结合，通过扩展 LCM 的基本原理来分离特定细胞类型，从而创建一种独立于操作员的方法来获取特定 CNS 细胞类型。结果我们报告了 xMD 如何能够从大鼠福尔马林固定石蜡包埋 (FFPE) 组织中分离特定细胞群，即神经元和星形胶质细胞。随后的逆转录酶聚合酶链反应（RT-PCR）分析证实了这些特定群体的富集。 xMD 后的 RIN 值表明样品足以进行进一步分析。与现有方法相比，xMD 提供了一种快速分离特定 CNS 细胞类型的方法，无需操作员进行识别，减少了操作员错误造成的无意污染量，同时还显着减少了当前基本 LCM 技术所需的时间。结论 xMD 是从死后组织中获取富集细胞群的优越方法，可用于创建转录组图谱，有助于我们了解其贡献这些细胞导致一系列神经系统疾病。 xMD 还解决了与 LCM 相关的问题，例如依赖操作员识别目标细胞（这可能会导致污染），以及解决 LCM 耗时的问题。
BackgroundLaser capture microdissection (LCM) is an established technique for the procurement of enriched cell populations that can undergo further downstream analysis, although it does have limitations. Expression microdissection (xMD) is a new technique that begins to address these pitfalls, such as operator dependence and contamination.New methodxMD utilises immunohistochemistry in conjunction with a chromogen to isolate specific cell types by extending the fundamental principles of LCM to create an operator-independent method for the procurement of specific CNS cell types.ResultsWe report how xMD enables the isolation of specific cell populations, namely neurones and astrocytes, from rat formalin fixed-paraffin embedded (FFPE) tissue. Subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirms the enrichment of these specific populations. RIN values after xMD indicate samples are sufficient to carry out further analysis.Comparison with existing methodxMD offers a rapid method of isolating specific CNS cell types without the need for identification by an operator, reducing the amount of unintentional contamination caused by operator error, whilst also significantly reducing the time required by the current basic LCM technique.ConclusionsxMD is a superior method for the procurement of enriched cell populations from post-mortem tissue, which can be utilised to create transcriptome profiles, aiding our understanding of the contribution of these cells to a range of neurological diseases. xMD also addresses the issues associated with LCM, such as reliance on an operator to identify target cells, which can cause contamination, as well as addressing the time consuming nature of LCM.