Appleby-Mallinder C, Wyles M D, Simpson J E, Wharton S B, Ince P G, Heath P R
Sheffield Institute for Translational Neuroscience, University of Sheffield, UK.
Sheffield Institute for Translational Neuroscience, University of Sheffield, UK.
J Neurosci Methods. 2016 Aug 1;268:125-30. doi: 10.1016/j.jneumeth.2016.05.007. Epub 2016 May 10.
Laser 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.
xMD 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.
We 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.
xMD 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.
xMD 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.
激光捕获显微切割（LCM）是一种用于获取可进行进一步下游分析的富集细胞群体的成熟技术，尽管它确实存在局限性。表达显微切割（xMD）是一种新出现的技术，开始解决这些问题，如对操作人员的依赖和污染问题。
xMD通过扩展LCM的基本原理，利用免疫组织化学结合显色剂来分离特定细胞类型，从而创建一种无需操作人员的方法来获取特定的中枢神经系统细胞类型。
我们报告了xMD如何从大鼠福尔马林固定石蜡包埋（FFPE）组织中分离出特定的细胞群体，即神经元和星形胶质细胞。随后的逆转录聚合酶链反应（RT-PCR）分析证实了这些特定群体的富集。xMD后的RNA完整性数（RIN）值表明样本足以进行进一步分析。
xMD提供了一种快速分离特定中枢神经系统细胞类型的方法，无需操作人员进行识别，减少了因操作失误导致的无意污染量，同时也显著减少了当前基本LCM技术所需的时间。
xMD是一种从死后组织中获取富集细胞群体的优越方法，可用于创建转录组图谱，有助于我们了解这些细胞对一系列神经系统疾病的作用。xMD还解决了与LCM相关的问题，如依赖操作人员识别靶细胞可能导致污染，以及LCM耗时的问题。
