Session 6: Emerging Technologies
C. Huang
Autism may involve multiple pathogenic mechanisms acting on a number of cellular targets in the brain. At the molecular level, a collection of biomarkers rather than a single gene or protein may be required to better define the detailed mechanisms of this complex disorder. Methods of cell-specific extraction from heterogeneous tissue offer an attractive starting point for the analyses of DNA, mRNA, and proteins. Laser capture microdissection (LCM) is a recent addition to a number of microdissection procedures aimed to procure pure populations of cells for subsequent molecular analysis. Emmert-Buck et al [1996] and Bonner et al [1997] have introduced the use of near-infrared laser to heat and melt a thermoplastic polymer film in order to lift the selected cells for subsequent analysis. Boehm et al [1997] have used UV laser beam to cut out the tissue of interest. Microdissections can be carried out on frozen sections as well as paraffin-embedded sections (surgical and autopsy), including those prepared from formalin- or ethanol-fixed tissue. Areas in the 30- to 700mm diameter range are being dissected in a routine manner whereas smaller areas (5-10mm) are also feasible.
Under optimal laboratory conditions, mRNA isolated from as few as 200 cells resulted in detection of low, medium, and highly expressed genes by RT-PCR [Dolter and Braman 2001]. Proteomic analysis, however, currently still requires the collection of 100,000 cells or more [Hanash 2001]. In the near future, laser capture microdissection techniques are expected to see rapid progress, resulting in further improvement in automation as well as the purity of the dissected cellular sample. These advances, coupled with the expected progress in the improved sensitivity of mRNA and proteomic analysis, should bring significant insights to the underlying pathological processes leading to autism.
References
- Boehm M et al. (1997) Am J Pathol 151: 63.
- Bonner RF et al. (1997) Science 278: 1481.
- Dolter KE, Braman JC (2001) Biotechniques 30:1358.
- Emmert-Buck et al. (1996) Science 274:998.
- Hanash S (2001) Proceedings of the Conference on Laser Capture Microdissection and Macromolecular Analysis of Normal Development and Pathology, July 17-18, 2001, Bethesda, MD.
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