Microdissected cells

Johnson NA, Hamoudi RA, Ichimura K, et al. Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells. Lab. Invest. 2006 86 968-978. 

Batorfi, J., Ye, B., Mok, S.C., Cseh, I., Berkowitz, R.S., and Fulop, V., Protein profiling of complete mole and normal placenta using ProteinChip analysis of laser capture microdissected cells, Gynecol. Oncol., 88, 424-428, 2003. 

Xu BJ, Caprioli RM, Sanders ME, Jensen RA. Direct analysis of laser capture microdissected cells by MALDI mass spectrometry. J Am Soc Mass Spectrom 2002 13(11) 1292-1297. 

Example of Use Analysis of 1000 Microdissected Cells from PanIN Grades for the Identification of a New Molecular Tumor Marker Using CyDye DICE Saturation Fluors... 

In order to identify new molecular markers of the pancreatic tumor progression we established a proteomics approach analyzing 1000 microdissected cells from different pancreatic intraepithelial neoplasias (PanIN) grades (Sitek et al., 2005b). 

Figure 3.9 DICE analysis of pancreatic ductal epithelium and PanlN-2 microdissected cells. The analysis of 1000 microdissected cells from pancreatic ductal epithelium and PanlN-2 lesions revealed a pattern of A 1.875 and B 2.050 protein spots, respectively.

Table 3.3 First candidate molecular markers for PanlN-2 grade pancreas carcinoma analyzing 1000 microdissected cells.

The primary components of LCM technology are (1) visualization of the cells of interest through microscopy, (2) transfer of near-infrared laser energy pulses to a thermolabile polymer with formation of a polymer-cell composite, and (3) removal of the polymer from the tissue surface, which shears the embedded cells of interest away from the heterogeneous tissue section (18,19). Extraction buffers applied to the polymer film solubilize the cells, liberating the molecules of interest. The DNA, RNA, or protein from the microdissected cells may be analyzed by any method with appropriate sensitivity (20,21,22,23,24). Protein extracted from microdissected cells may be used for mass spectrometric analysis, applied to reverse phase protein microarrays, or used for western blot analysis (25,26). 

Microdissected cells for protein analysis may be stored at -80°C before extraction. Microdissected cells for DNA analysis may be stored desiccated at room temperature up to 1 week before extraction. Samples for RNA analysis should not be stored before extraction. Condensation in the microcentrifuge tube during storage may be a potential source of RNAse contamination. RNA extraction should be performed immediately after microdissection. Store extracted RNA samples at -80°C before amplification. 

Optimal tissue thickness for microdissection is 5-8 pm. Tissue sections less than 5 pm may not provide a full cell thickness, necessitating microdissection of more cells for a given assay. Tissue sections thicker than 8 pm may not microdissect completely, leaving integral cellular components adhering to the slide. Ethanol fixed tissue sections may also be used for proteomic analysis, but a larger number of microdissected cells may be required for adequate sensitivity in downstream assays as compared with frozen sections. 

Laser capture microdissection was combined with MALDl-MS by several groups [49-52], In one study, the MALDl analysis of microdissected cells from human breast cancer in comparison to normal breast epithelial cells was reported [49], with more than 40 peaks in the MALDl mass spectra being found to differ in intensity between the cancer and normal tissues. This approach represents another way of achieving laterally resolved analytical information. In this case, it was not the focus size of the desorbing laser beam in the mass spectrometer. 

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