Imamichi Yukiko, Koebernick Katja, Wedlich Doris
Department of Internal Medicine I, University of Ulm, Germany.
Pathol Res Pract. 2003;199(6):381-9. doi: 10.1078/0344-0338-00434.
Analysis of spatiotemporal patterns of gene expression is an important prerequisite for understanding the molecular basis of embryogenesis. Tissue-specific resolution is desirable, but often not achieved owing to methodical limitations. We used a common model system for embryonic development--the South African clawed frog Xenopus laevis--to demonstrate that laser microdissection and laser-mediated catapulting of tissue samples from histologic sections are feasible even for yolk-rich, fragile embryonic tissue. A combination with RT-PCR provides the possibility of detecting tissue-specific gene expression with high resolution and fidelity. We show that specimens of various sizes and shapes can easily be procured by laser microdissection and pressure catapulting (LMPC). Subsequent RNA-isolation and nested RT-PCR for marker genes revealed that the combination of these methods allows for analysis of specific gene expression in micro-areas. We report on the efficiency and reliability of detection of marker genes in dissected tissue. We further discuss the question of whether such a combination can be applied to certain issues raised in developmental biology with regard to other techniques.
分析基因表达的时空模式是理解胚胎发育分子基础的重要前提。理想情况下需要组织特异性分辨率，但由于方法上的局限性往往难以实现。我们使用了一种常见的胚胎发育模型系统——南非爪蟾非洲爪蟾，来证明从组织学切片中进行激光显微切割和激光介导的组织样本弹射，即使对于富含卵黄、脆弱的胚胎组织也是可行的。与逆转录聚合酶链反应（RT-PCR）相结合，提供了以高分辨率和保真度检测组织特异性基因表达的可能性。我们表明，通过激光显微切割和压力弹射（LMPC）可以轻松获取各种大小和形状的样本。随后对标记基因进行RNA分离和巢式RT-PCR，结果显示这些方法的组合能够分析微区域中的特定基因表达。我们报告了在解剖组织中检测标记基因的效率和可靠性。我们还进一步讨论了这种组合是否可以应用于发育生物学中与其他技术相关的某些问题。
