Abstract
Laser-assisted microdissection has enabled the collection of morphologically defined cell populations from a tissue section. The PALM® Robot MicroBeam laser microdissection system provides a robust system for the retrieval of specified cells (including single cells). Due to the fragile nature of DNA, and in particular RNA, robust protocols are required to obtain reliable data from a limited number of cells (1-10.000 cells). This chapter describes the application of the PALM MicroBeam system to isolate RNA and DNA from cells in a complex tissue for subsequent molecular analysis. Protocols for successful analysis of RNA from 500 to 1000 cells, including steps to produce cDNA for subsequent polymerase chain reaction analysis, are given. The cDNA could also be used as a template for linear amplification in order to perform gene array analysis. Furthermore, a protocol for genomic analysis of p53 mutations from single cells is given. The described procedures emphasize preparation of tissue, laser microdissection including catapulting of cells, and extraction of RNA and DNA. Downstream experiments for validation are also shown.
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References
Schutze, K., Burgemeister, R., Clemment-Sengewald, A., Ehnle, S., Friedemann, G., Lahr, G., et al. (2003) Non-contact live cell laser micromanipulation using PALM microlaser systems. P.A.L.M Scientific Edition, 11.
Schutze, K., Posl, H., and Lahr, G. (1998) Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine. Cell. Mol. Biol. (Noisy-le-grand) 44, 735–746.
Williams, C., Ponten, F., Moberg, C., Soderkvist, P., Uhlen, M., Ponten, J, et al. (1999) A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am. J. Pathol. 155, 1467–1471.
Micke, P., Bjørnsen, T., Scheidl, S., Strömberg, S., Demoulin, J. B., Ponten, F., et al. (2004) A fluid cover medium provides superior morphology and preserves 166 RNA integrity in tissue sections for laser microdissection and pressure catapulting. J. Pathol. 202, 130–138.
Scheidl, S. J., Nilsson, S., Kalen, M., Hellstrom, M., Takemoto, M., Hakansson, J., and Lindahl, P. (2002) mRNA expression profiling of laser microbeam micro-dissected cells from slender embryonic structures. Am. J. Pathol. 160, 801–813.
Persson, Å., Ling, G., Williams, C., Bäckvall, H., Ponten, J., Ponten, F., and Lundeberg, J. (2000) Efficient analysis of single cells obtained from histological tissue sections. Anal. Biochem. 287, 25–31.
Ling, G., Persson, Å., Berne, B., Uhlen, M., Lundeberg, J., and Ponten, F. (2001) Persistent p53 mutations in single cells from normal human skin. Am. J. Pathol. 159, 1247–1253.
Wester, K., Asplund, A., Backvall, H., Micke, P., Derveniece, A., Hartmane, I., et al. (2003) Zinc-based fixative improves preservation of genomic DNA and proteins in histoprocessing of human tissues. Lab. Invest. 83, 889–899.
Vincek, V., Nassiri, M., Knowles, J,. Nadji, M., and Morales, A. R. (2003) Preservation of tissue RNA in normal saline. Lab. Invest. 83, 137–138.
Lehmann, U. and Kreipe, H. (2001) Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 25, 409–418.
Becker, I., Becker, K. F., Rohrl, M. H., and Hofler, H. (1997) Laser-assisted preparation of single cells from stained histological slides for gene analysis. Histochem. CellBiol. 108, 447–451.
Specht, K., Richter, T., Muller, U., Walch, A., Werner, M., and Hofler, H. (2001) Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am. J. Pathol. 158, 419–429.
Burgemeister, R., Gangnus, R., Haar, B., Schutze, K., and Sauer, U. (2003) High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology. Pathol. Res. Pract. 199, 431–436.
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Micke, P., Östman, A., Lundeberg, J., Ponten, F. (2005). Laser-Assisted Cell Microdissection Using the PALM System. In: Murray, G.I., Curran, S. (eds) Laser Capture Microdissection. Methods in Molecular Biology™, vol 293. Humana Press. https://doi.org/10.1385/1-59259-853-6:151
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DOI: https://doi.org/10.1385/1-59259-853-6:151
Publisher Name: Humana Press
Print ISBN: 978-1-58829-260-5
Online ISBN: 978-1-59259-853-3
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