Summary
One of the major challenges in molecular analysis of breast cancer specimens is tissue heterogeneity. The admixture of contaminating bystander cells might distort the results of quantitative molecular analyses. Therefore, pure tumor cell populations have to be isolated in order to obtain reliable molecular data.
In this chapter, we present protocols for the laser-assisted microdissection of breast cancer tissue sections (using a laser microdissection microscope from P.A.L.M.®, Bernried, Germany) and the subsequent isolation of genomic DNA or total RNA. The protocols presented in here have been used in our laboratory for the exact quantification of gene copy numbers in intraductal and invasive tumor cells and for the quantitative assessment of promoter hypermethylation during breast cancer progression.
We have added some guidelines for the organization of the laser-microdissection and polymerase chain reaction laboratory, which prevent crosscontamination of samples and carry-over contamination because of polymerase chain reaction products.
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Suggested Readings
Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., et al. (1996) Laser capture microdissection. Science 274, 998–1001, and Simone, N. L., Bonner, R. F., Gillespie, J. W., Emmert-Buck, M. R., and Liotta, L. A. (1998) Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet. 14, 272–276.
Current Protocols of Molecular Biology (Ausubel et al., eds. Wiley, New York, NY).
Fink, L., Seeger, W., Ermert, L., et al. (1998) Real-time quantitative RT-PCR after laser-assisted cell picking. Nat. Med. 4, 1329–1333.
Luo, L., Salunga, R. C., Guo, H., et al. (1999) Gene expression profiles of lasercaptured adjacent neuronal subtypes (published erratum appears in Nat. Med. [1999] 5, 355). Nat. Med. 5, 117–122.
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Lehmann, U., Kreipe, H. (2006). Laser-Assisted Microdissection and Isolation of DNA and RNA. In: Brooks, S.A., Harris, A. (eds) Breast Cancer Research Protocols. Methods in Molecular Medicine, vol 120. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-969-9:65
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DOI: https://doi.org/10.1385/1-59259-969-9:65
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-191-2
Online ISBN: 978-1-59259-969-1
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