An optimized protocol for Laser Capture Micro-dissection for cytogenetic characterization of colon cancer specimens

A late diagnosis of colorectal cancer leads to a significant reduction of average survival time. In current screening programs most of the tumours are detected at advanced stages. Therefore, highly sensitive and specific biomarkers must be identified to allow malignancy detection at very early stages. Colon cancer specimens consist of heterogeneous tissue including different cell types such as tumour-, inflammatory- and stroma-cells. For a better understanding of cancer tumourgenesis and to identify potential biomarkers of high sensitivity and specificity, it is thus crucial to dissect uncontaminated cell types for further analyses. The method of Laser Capture Micro-dissection (LCM) allows the selection and dissection of e.g., tumour cells independent and free of surrounding cells. The drawbacks of LCM however are the time-excessive workload and the low yield of DNA-, RNA- and protein of the dissected cells. In order to address these limitations of LCM we aimed at identifying an optimized workflow to gain a high DNA- and RNA-yield of high quality with decent workload and decreased time investment. Thus, colorectal cancer samples and non-aberrant, healthy colon samples were prepared for kryo-sectioning of different thickness (e.g., 12, 25 and 30 micrometer) under RNase- and Dnase-free conditions. The sections were placed on special membrane-slides and stained with toluidin-blue for subsequent micro-dissection. The dissected cells were further examined by different extraction methods for RNA and DNA (classical method versus column-assisted-method). Hereby, we evaluated the minimum input of cells (area) in order to perform cytogenetic analyses (m-arrays and arrayCGH) without any further amplification of the yielded RNA/DNA. The isolated RNA and DNA are highly representative for the transcriptome and genome of the cell type of interest. An optimized workflow for LCM on kryo-sections will be presented providing the basis for a more specific genomic and transcriptomic analysis of distinct cell types, that allows the extraction of highly cell type-representative DNA and RNA of high yield and quality with limited time investment for biomarker screening at a manifold increased resolution and potentially provides new insights into colorectal carcinogenesis.