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Laser capture microdissection: understanding the techniques and implications for molecular biology in nursing
Karen L Zanni1, Garrett K Chan
1Binghamton University, Decker School of Nursing, Binghamton, NY, Summer Genetics Institute, National Institute of Nursing Research, Bethesda, MD, USA.
Aim:
The purpose of this paper is to review the techniques and implications of laser capture microdissection (LCM) to isolate tissue and DNA of interest using breast biopsy tissue as an example.
Background:
Tissues are a heterogeneous mix of different cell types, and molecular alterations are often specific to a single cell type. An accurate correlation of molecular and morphologic pathologies requires the ability to procure pure populations of morphologically similar cells for molecular analysis. LCM is a technique for isolating highly pure cell populations of morphologically similar cells from a heterogeneous tissue section.
Method:
Nine invasive, paraffin-embedded breast biopsy specimens were obtained and analyzed. Depending on the size of the lesion, 500-1,000 shots using the 7.5- or 15-µm infrared laser beam were utilized to obtain an average of 2,000 cells. DNA was isolated from normal tissue and carcinomas and polymerase chain reaction (PCR) amplification was examined by agarose gel electrophoresis. The HER2/neu gene was amplified by standard PCR. A second round of PCR using nested primers to re-amplify the HER2/neu fragment was performed.
Results:
Amplification of the HER2/neu gene with DNA isolated from pure cell populations by LCM was performed. The results indicated that 22% of the cases studied were positive for HER2/neu amplifications, which corresponds to the literature regarding HER2/neu amplification/overexpression. HER2/neu amplification could be detected as early as the ductal carcinoma in situ (DCIS) stage.
Conclusion:
LCM is an accurate and reliable method to acquire nucleic acid and protein profiles from a specific cell population in heterogeneous tissue.
