Morrison Jason A, Bailey Caleb M, Kulesa Paul M
Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
Cold Spring Harb Protoc. 2012 Dec 1;2012(12):pdb.prot072140. doi: 10.1101/pdb.prot072140.
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.
发育中胚胎的动态特性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具有挑战性。更具洞察力的是在特定发育阶段对少量细胞进行选择性检测的分子谱分析。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仍然是一项重大挑战。在此，我们描述了使用激光捕获显微切割（LCM）、免疫组织化学（IHC）和逆转录定量聚合酶链反应（RT-qPCR），以比任何其他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，通过LCM特异性识别用于收获的细胞，而不会导致总RNA的显著降解。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RT-qPCR结果。基因特异性预扩增仅从收获的材料中高效且线性地扩增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RT-qPCR相结合，我们能够可靠地分析从LCM收获的低至10个细胞中约300个基因的表达。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，克服了限制基因谱分析的技术障碍。
