Amplification testing in breast cancer by multiplex ligation-dependent probe amplification of microdissected tissue

This chapter describes a method for the rapid assessment of gene copy numbers in laser-microdissected materials using multiplex ligation-dependent probe amplification (MLPA). An MLPA is a powerful multiplex PCR technique that can identify gains, amplification, or losses of up to 50 genes in a single experiment, thereby requiring only minute quantities of DNA extracted from frozen or paraffin-embedded materials. A previous study in breast cancer has shown that MLPA can detect amplifications in cases with a tumor percentage lower than 10%, but still a low tumor percentage in the tissue tested could obscure low levels of amplification due to dilution of the tumor cell population by normal cells. Laser capture microdissection allows enrichment of tumor cells by eliminating background noise from normal and preinvasive cells, thereby increasing specificity and sensitivity.This chapter describes a method for MLPA analysis using invasive breast tumor cells acquired by laser capture microdissection. This protocol can also be applied to MLPA analysis of preinvasive lesions and metastases.