Combination of microdissection and microarray analysis to identify gene expression changes between differentially located tumour cells in breast cancer.

Abstract

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100-200 ng, from this microdissected tissue required use of the Atlas SMART trade mark Probe Amplification Kit to synthesize and amplify cDNA and make (33)P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.