Laser-assisted microdissection and isolation of DNA and RNA.

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.