Visualization and Isolation of Human Notochord: An Approach Using Laser Capture Microdissection

Background: The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. Therefore, the notochord seems to be the most appropriate control tissue in chordoma gene expression studies. Many studies have used different tissues as a control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. Methods: Histological examination of three FFPE spines of human fetuses, with a gestation between 9 and 13 weeks, using (immuno)histochemical methods was performed. Dissection of the notochord from a flash frozen fetus of 10 weeks gestation was performed using laser capture microdissection (LCM). RNA was extracted from this tissue and used in quantitative PCR. Results: This study illustrates notochord of embryonic spines in three different stages of gestation (9-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of the notochord and subsequent RNA extraction resulted in a good yield of total RNA. qPCR analysis confirmed the high quality of RNA. Conclusion: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis.