Microdissection

Fig. 2. Methods for production of identical catde. (a) Embryo spHtting. A microdissection knife is used to cut an early embryo into two identical halves, (b) Embryo cloning. A cell from a morula stage embryo is removed and then inserted into an oocyte that has had its nucleus removed. EoUowing fusion of the two cells the newly produced one-ceU embryo is identical to the original morula. The procedure can be repeated to give multiple copies of the original...

Torres-Munoz JE, Nunez M, Petito CK (2008) Successful Application of Hyperbranched Multidisplacement Genomic Amplification to Detect HlV-1 Sequences in Single Neurons Removed from Autopsy Brain Sections by Laser Capture Microdissection. J Mol Diagn 10(4) 317-324... 

Kilpatrick, L Jones, M. and Phillipson, O. A semiautomated analysis method for catecholamines, indoleamines, and some prominent metabolites in microdissected regions of the nervous system An isocratic HPLC technique employing eoulometric detection and minimal sample preparation. J Neurochem 46(6) 1865-1876, 1986. 

Siezen, R. J., Kaplan, E. D., and Anello, R. D., Superior resolution of y-crystal-lins from microdissected eye lens by cation-exchange high-performance liquid chromatography, Biochem. Biophys. Res. Comm., 127, 153, 1985. 

Ornstein DK et al. Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines. Electrophoresis 2000 21 2235-2242. 

Volume 356. Laser Capture Microscopy and Microdissection Edited by P. Michael Conn... 

Lehmann et al.58 Microdissected CD68 (+) cell from liver section N/A Real time, for oncogenes with amplicon size of 150-300 bp It may provide a powerful tool to study gene alterations in FFPE tissue. 

Lahr et al.59 Microdissected thyroid tissues PUREscript kit (BlOzym, Germany) Nested PCR, RET oncogene with amplicon size of 141-383 bp Expressed genes can be analyzed from routine FFPE tissue slides or pooled single cells. 

Specht et al.60 Microdissected cancer tissues RNA lysis buffer containing SDS and proteinase K Real time, for seven cancerrelevant genes Reproducible quantitation of specific mRNAs can be achieved... 

Jin et al.64 Microdissected TRIzol reagent 20 specific chimeric Positive signal... 

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... 

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. 

Stoecklein NH, Erbersdobler A, Schmidt-Kittler O, et al. SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples. Am. J. Pathol. 2002 161 43-51. 

Lehmann U, Bock O, Glockner S, et al. Quantitative molecular analysis of laser-microdissected paraffin-embedded human tissues. Pathobiology 2000 68 202-208. 

Lahr G, Sticha M, Schtitzea K, et al. Diagnosis of papillary thyroid carcinoma is facilitated by using an RT-PCR approach on laser-microdissected archival material to detect RET oncogene activation. Pathobiology 2000 68 218-226. 

Specht K, Richter T, Muller U, et al. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embeddded tumor tissue. Am. J. Pathol. 2001 158 419-429. 

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. 

Lindeman N, Waltregny D, Signoretti S, et al. Gene transcript quantitation by real-time RT-PCR in cells selected by immunohistochemistry-laser capture microdissection. Diagn. Mol. Pathol. 2002 11 187-192. 

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. 

Guo T, Wang W, Rudnick PA, et al. Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens. J. Histochem. Cytochem. 2007 55 763-772. 

A number of proteomic studies on archival material have utilized Liquid Tissue (Expression Pathology, Inc., Gaithersburg, MD), a commercial protein extraction kit for FFPE tissue.4,9,25-28 This kit is also based upon HIAR techniques and shares a similar work flow to the methods already discussed. Thin, typically 5-10pM, sections are cut from paraffin tissue blocks, the paraffin is removed, and the tissue deparaffinized and rehydrated in alcohols and distilled water before microdissection. The cellular material is then suspended in Liquid Tissue buffer and heated at 95°C for 90 min. Trypsin is added, and the material is digested overnight at 37°C prior to reduction with DTT and analysis by LC-MS/MS.26... 

EVALUATION OF LASER CAPTURE MICRODISSECTION OF FFPE TISSUES... 

The proteins uniquely identified in the whole section corresponded to proteins typically found in blood or associated with the extracellular matrix. LCM effectively minimized the presence of these proteins in the microdissected sample. The enrichment effect is better illustrated by comparing the relative quantities of proteins identified in each sample (Fig. 20.2). 

Figure 20.1 Venn diagram of proteins identified from microdissected tissue (left) versus non-microdissected tissue (right).

SHOTGUN PROTEOME ANALYSIS OF MICRODISSECTED FORMALIN-FIXED BRAIN TUMOR TISSUE... 

In one experiment, using the AR-based protocol for protein extraction from microdissected FFPE human tissue section, a total of 14,478 distinct... 

In 2004, Melle et al.12 proposed a technical triad of microdissection, pro-teomic techniques, and IHC that provides an integrated approach for characterization of cancer biomarkers. In recent years, a growing body of literature pertaining to this combined approach has accumulated. Table 22.1 shows a few selected examples under this topic. 

LCM, laser captured microdissection ESI, electrospray ionization PAGE, polyacrylamide gel electrophoresis IGF-1R, insulin-like growth factor receptor 1. 

---