Atkins Norman, Miller Charlie M, Owens Joseph R, Turek Fred W
Center for Sleep and Circadian Biology, Northwestern University.
J Vis Exp. 2011 Nov 13(57):3125. doi: 10.3791/3125.
As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)(1) provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs(2-4). Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling(5) within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression(6). However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies. Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision(7,8). While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-μm serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.
作为技术平台，诸如新一代测序、微阵列和定量逆转录聚合酶链反应等方法在扩展我们对分子调控广度的理解方面具有巨大潜力。诸如高分辨率RNA测序（RNA-Seq）（1）等更新的方法提供了有关组织或状态特异性表达的新的广泛信息，如相对转录水平、可变剪接和微小RNA（2-4）。在离散组织内或表型不同的个体组之间采用RNA-Seq方法进行比较全转录组分析（5）的前景为阐明正常和异常生理状态下的分子机制提供了新途径。最近，已经对人类脑组织进行了全转录组分析，确定了与疾病进展相关的基因表达差异（6）。然而，新一代测序的应用尚未更广泛地整合到哺乳动物研究中。在小鼠模型中的基因表达研究报告了使用激光捕获显微镜（LCM）进行样本切除后在各种脑核内的不同表达谱（7,8）。虽然LCM能够以单细胞和离散脑区的精度进行样本采集，但LCM方法产生的相对较低的总RNA产量可能会限制其在小鼠脑组织中的RNA-Seq和其他分析方法的应用，并且可能需要次优的样本扩增步骤。在此，我们介绍一种从离散的小鼠脑区进行显微切割和总RNA提取的方案。使用固定直径的组织取芯器从一只小鼠脑的750μm连续冠状切片中分离出13个组织。组织微打孔样本立即冷冻并存档。使用基于磁珠的总RNA分离技术从样本中获得总RNA。所得RNA样本具有足够的产量和质量，可用于下游表达分析。这种显微切割策略为从离散脑区获取总RNA的现有样本采集策略提供了一个可行的选择，为新的基因表达发现开辟了可能性。
