Improved RNA preservation for immunolabeling and laser microdissection
Improved RNA preservation for immunolabeling and laser microdissection
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DOI：
10.1261/rna.1733509
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发表时间：
2009-12-01
期刊：
影响因子：
4.5
通讯作者：
Smith, Doug W.
中科院分区：
文献类型：
--
作者：
Brown, Amanda L.;Smith, Doug W.
显微切割技术有可能允许从异质组织（如神经系统和某些癌症）中分离的特定细胞群中进行转录组分析。问题是，RNA在通常需要鉴定用于显微切割的感兴趣细胞的标记条件下不稳定。我们已经开发了一种免疫标记方法，该方法利用高盐缓冲液在长时间的抗体孵育期间稳定RNA。我们首先通过三种方法评估了RNA的完整性，发现在高盐缓冲液中孵育至少20小时的组织产生的RNA质量与从新鲜冷冻组织中提取的RNA质量相似，这被认为是最高质量的。值得注意的是，其完整性上级使用快速免疫标记程序（总持续时间5分钟）处理的组织中提取的RNA。我们接下来确定了高盐缓冲液与免疫标记相容，如通过脑中多巴胺神经元的免疫荧光检测所证明的。最后，我们用激光显微切割多巴胺神经元，用高盐缓冲液免疫标记，并证明RNA的完整性得到保留。我们所描述的方法产生高质量的RNA从免疫标记的显微切割细胞，一个有意义的基因组学研究从复杂的组织中分离的正常和病理细胞的基本要求。
Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.