By Graeme I. Murray, Stephanie Curran
Professional laboratorians famous for his or her optimized equipment describe in step by step aspect find out how to effectively perform tissue-based laser microdissection of their personal laboratory utilizing the several platforms on hand and to use quite a lot of refined molecular applied sciences to the microdissected tissue samples. Highlights comprise gene expression profiling of basic tumor mobilephone populations utilizing laser trap microdissection, RNA transcript amplification and GeneChip® microarrays, laser seize microdissection and colorectal melanoma proteomics, and proteomic research of human bladder tissue utilizing SELDI® technique following microdissection recommendations. functions of the microdissected tissue defined surround PCR, gene expression experiences together with microarray research, and proteomics.
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Additional info for Laser Capture Microdissection: Methods and Protocols
M. (1970) Methacarn (Methanol-Carnoy) fixation. Practical and theoretical considerations. Histochemie 21, 97–116. 11. , and Gusterson, B. A. (1985) Improved immunohistochemical localization of tissue antigens using modified methacarn fixation. J. Histochem. Cytochem. 33, 491–495. 12. , et al. (1999) Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am. J. Pathol. 154, 83–95. 13. , and Eimoto, T. (2000) Influence of histochemical and immunohistochemical stains on polymerase chain reaction.
To amplify target fragment sizes smaller than 1 kb, PCR was performed with cycle parameters of 95°C for 5 min, 35 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 30 s. 5 min respectively. Although the source of cells and the detection system are different from those in the present study, similar performance was obtained when DNA from 25 cells of alcohol-fixed cytology specimens was used in the multiplex PCR (27). Acknowledgments This work was supported in part by Health and Labour Sciences Research Grants (Risk Analysis Research on Food and Pharmaceuticals) from the Ministry of Health, Labour, and Welfare of Japan.
This chapter outlines the techniques involved in producing labeled probes from DNA extracted from laser capture microdissected material and the methods for hybridization of these probes to metaphase chromosomes. This protocol can also be applied to the preparation of probes for CGH arrays. Key Words: Laser capture microdissection (LCM); comparative genomic hybridization (CGH); degenerate oligonucleotide primed PCR (DOP-PCR); array CGH. 1. Introduction Comparative genomic hybridization (CGH) is a powerful whole-genome screening technique that allows analysis of DNA copy-number losses (deletions) and gains across the whole genome in a single experiment.