Gene profiling in the avian embryo using laser capture microdissection and RT-qPCR.
Gene profiling in the avian embryo using laser capture microdissection and RT-qPCR.
复制标题
DOI：
10.1101/pdb.prot072140
复制
发表时间：
2012-12
影响因子：
--
通讯作者：
Jason A. Morrison;C. Bailey;P. Kulesa
中科院分区：
文献类型：
--
作者：
Jason A. Morrison;C. Bailey;P. Kulesa
发育中的胚胎具有动态特性，这使得利用大规模基因表达模式的信息来理解复杂的形态发生事件颇具挑战性。更有洞察力的做法是对在特定发育阶段有选择地检测的少量细胞进行分子分析。然而，从同质组织中的少量细胞（<10个）中检测基因表达谱信息一直是一项重大挑战。在此，我们描述了利用激光捕获显微切割（LCM）、免疫组织化学（IHC）和实时定量聚合酶链反应（RT - qPCR）来比其他任何方法都更精确地提取不同胚胎组织中的基因谱信息。我们使用鸡胚模型系统，并结合电穿孔和双标记免疫组织化学来特异性地识别可供激光捕获显微切割采集的细胞，同时总RNA不会出现明显降解。我们描述了一种针对小细胞亚群的预扩增方案的开发，以产生灵敏的实时定量聚合酶链反应结果。基因特异性预扩增能够有效且线性地仅扩增所采集材料中感兴趣的基因转录本，而无需进行RNA分离。通过将上述技术与微流控实时定量聚合酶链反应相结合，我们能够可靠地分析从激光捕获显微切割采集的少至10个细胞中约300个基因的表达情况。总之，该方案提供了一种可靠的分离方法以及对组织中少量细胞进行灵敏表达分析的手段，克服了限制基因分析的一个技术障碍。
The dynamic nature of the developing embryo makes it challenging to understand complex morphogenetic events using information from large-scale gene expression patterns. What would be more insightful is molecular profiling of small numbers of cells selectively surveyed at specific developmental stages. However, detecting gene expression profile information from small numbers of cells (<10) in homogenous tissue has remained a major challenge. Here, we describe the use of laser capture microdissection (LCM), immunohistochemistry (IHC), and RT-qPCR to extract gene profile information in distinct embryo tissue more precisely than is possible with any other method. We use the chick embryo model system and combine electroporation and dual-label IHC to specifically identify cells for harvest by LCM without significant degradation of total RNA. We describe the development of a pre-amplification protocol for small subpopulations of cells to produce sensitive RT-qPCR results. The gene-specific pre-amplification efficiently and linearly amplifies only gene transcripts of interest from the harvested material without the need for RNA isolation. By combining the above techniques with microfluidic RT-qPCR, we robustly analyze the expression of ∼300 genes from as few as 10 cells harvested by LCM. Together, this protocol presents a confident isolation and means of sensitive expression analysis of small cell numbers from tissues and overcomes a technical hurdle that limits gene profiling.