Laser capture microdissected tissue

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

An alternative approach to assessing tissue-specific expression at the proteomic level can be achieved by MS of laser capture microdissected tissues.4 An important development in this arena is the ability to perform LCM and MS/MS on formalin-fixed paraffin-embedded tissues. 

Pahner-Toy, D.E., Sarracino, D.A., Sgroi, D., LeVangie, R. and Leopold, P.E., Direct acquisition of matrix-assisted laser desorption/ionization time-of-flight mass spectra from laser capture microdissected tissues. Clin. Chem., 46, 1513-1516 (2000). 

Numerous articles have demonstrated the availability of RNA extracted from a few cells or even a single cell taken by laser captured microdissection (LCM) system from archival FFPE tissue sections that had been previously stained by IFIC. Using a combination of pre-immunostained FFPE tissue section with LCM, a sensitive real-time quantitative RT-PCR can be achieved based on a few immuno-detected cells, creating a way to study pathophysiological gene regulation in a cell-specific manner in archival tissues housed in... 

Jin L, Majerus J, Oliveira A, et al. Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and RT-PCR. Diagn. Mol. Pathol. 2003 12 224-230. 

Tanji N, Ross MD, Cara A, et al. Effect of tissue processing on the ability to recover nucleic acid from specific renal tissue compartments by laser capture microdissection. Exp. Nephrol. 2001 9 229-234. 

EVALUATION OF LASER CAPTURE MICRODISSECTION OF FFPE TISSUES... 

During recent years, many of our appheations of competitive RT-PCR have involved the analysis of RNA from very small tissue samples obtained by laser capture microdissection. We have therefore adopted a modified reamphfication protocol (competitive Nested-RT-PCR) for the quantitation of very small tissue samples (Costa et al., 2002). The procedure involves the use of very low concentrations of cRNA (internal standard) in the presence of total RNA isolated from a measured and microdissected tissue sample or a... 

Paweletz CP, Liotta LA, Petricorn EE (2001) New technologies for biomarker analysis of prostate cancer progression laser capture microdissection and tissue proteomics. Urology 57 160-163... 

Craven BA, Totty N, Harnden P, Selby PJ, Banks RE. (2002) Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis evaluation of tissue preparation and sample limitations. Am J Pathol 160, 815-22. 

Microdissection of a tumor was initially carried out using a standard syringe (5). The tumor was viewed under a microscope so that it could be separated from the surrounding normal tissues. For this method to be effective, tumors needed to be easily defined under the microscope, which limited the number of samples that were compatible. Microdissection techniques have further evolved to include techniques such as laser microbeam microdissection (EMM) and laser capture microdissection (LCM). These... 

Suarez-Quian CA, Goldstein SR, Pohida T et al. Laser capture microdissection of single cells from complex tissues. Biotechniques 1999 26 328-335. 

In 2001, both Lawrie et al. and Moskaluk coupled LCM (laser capture microdissection) with 2D gel proteomics to recover proteins from laser captured micro-dissected tissue in a form that can be used in 2D gel analysis and mass spectrometry. This may provide valuable information for protein profiling and databasing of human tissues in healthy and disease states. 

At times there are very small amounts of tissue available for experimentation. Nevertheless, it is possible to isolate RNA from limited amounts of samples such as tumor biopsies or laser capture microdissected material [33], Although one can expect only small amounts of RNA from such tissues or cells, yields of RNA are enough for use in various applications, such as gene expression-related qualitative and quantitation studies using RT-PCR or even cDNA libraries [31,34], Small-scale RNA isolation is possible using commercially available kits that are available to isolate either total RNA or mRNA (Table 7.3). These kits can be used to isolate RNA from a few cells, or less than 1 mg of tissue. 

The importance for proteomic studies of being performed on standardized samples cannot be emphasized enough. Several approaches have been developed to address the problem of tissue heterogeneity. Beads-based sample enrichment (BBSE) and laser capture microdissection (LCM) procedures are emerging as the methods of choice. The aim of these procedures is to eliminate, as much as possible, all confounding and contaminating factors in order to obtain a sample quality that can be compared with cell lines without the disadvantages of in vitro cultures. 

Figure 16.2 2-D electrophoretic separations of laser capture microdissected human cardiac tissue.

Trillo-Pazos G, Diamanturos A, Rislove L, Menza T, Chao W, Belem P, Sadiq S, Morgello S, Sharer L, Volsky DJ (2003) Detection of HIV-1 DNA in microglia/macrophages, astrocytes and neurons isolated from brain tissue with HIV-1 encephalitis by laser capture microdissection. Brain Pathol 13 144-154. 

The ability to focus molecular expression analysis on only a limited number of cell types depends on cell separation methods that minimize the opportunity for other cell types to contribute to gene expression in situ. Even the most carefully gathered biological samples contain many cell types, especially if the sample is from inflamed or necrotic tissue. More homogeneous samples are provided by laser capture microdissection (LCM), a method that isolates individual cells or sections of tissue from a fixed sample [144-148]. The use of LCM minimizes contributions by nontarget cell populations in comparisons of diseased and normal tissues, but also introduces handling and preparation steps that can affect detection accuracy. 

Bonner RF, Emmert-Buck M, Cole K, Pohida T, Chuaqui R, Goldstein S, Liotta LA. Laser capture microdissection Molecular analysis of tissue. Science 1997 278(5342) 1481,1483. 

Paweletz, C.P Liotta, L.A. Petricoin, E.F. New Technologies for Biomarker Analysis of Prostate Cancer Progression Laser Capture Microdissection and Tissue Proteomics, Urology 57,160-163 (2001). 

The amount of tissue required for molecular testing depends on the sensitivity of the particular technique used and on the purity of the tumor sample (i.e., the proportion of tumor cells in a given specimen). Manual or laser capture microdissection can be used to enrich the tumor cell population. 

Su JM, Perlaky L, Li XN, Leung HC, Antalffy B, Armstrong D, Lau CC (2004) Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues. Brain Pathol 14 175-182... 

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