Questions Common To All Lcm Systems

Preparation of the specimen prior to LCM is an area in which the scientist should exercise great caution and care. Judging from the literature, the level of knowledge is in great flux, much as electron microscopy was in the 1950s. There are some very good standard references such as ref.6. However, even there, no mention is made of the potential for hematoxylin staining to affect the consistency of DNA template amplification by PCR, as reported by Gulley et al. (7). Specimen fixation definitely affects the quality of genetic material, with the consensus being that frozen specimens are best followed by alcohol fixation followed by formalin fixation (8). Possible effects on the quality of genetic material caused by laser irradiation or laser-generated heat during microdissection are in question. Different LCM manufacturers make different claims. Liotta et al. in the original description of LCM, in which a pulsed nitrogen laser (operating in the UV-A range of 320-400 nm) melted a thermoplastic film onto the tissue, found that the brief 90°C temperature exposure of the tissue had no effect on subsequent PCR analysis or enzyme assays of the microdissected specimen (3). Schütze et al. (9) claim no detrimental effects on RNA, DNA, or protein recovery from material catapulted using a UV-A laser.

The number of cells required for genetic analysis is generally in the range 300-500 cells for DNA and 500-1000 cells for RNA (10). For proteins, 1000-5000 cells are required when using gel analysis (11) and as few as 100 cells using SELDI mass spectroscopy (11).

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