Notochord isolation using laser capture microdissection

J Chem Neuroanat. 2017 Mar;80:37-43. doi: 10.1016/j.jchemneu.2016.12.004. Epub 2016 Dec 24.

Abstract

Background: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells.

Methods: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR.

Results: This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the 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. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.

Keywords: Chordoma; Human embryo; Laser-capture microdissection; Notochord; Tissue dissection.

MeSH terms

  • Female
  • Fetal Development
  • Fetus / anatomy & histology
  • Gestational Age
  • Humans
  • Immunohistochemistry
  • Laser Capture Microdissection / methods*
  • Notochord / anatomy & histology*
  • Pregnancy
  • RNA / biosynthesis
  • RNA / genetics
  • Real-Time Polymerase Chain Reaction
  • Spine / embryology

Substances

  • RNA