Laser Microdissection and RNA Analysis

  • Ludger Fink
  • Rainer Maria Bohle
Part of the Methods in Molecular Biology™ book series (MIMB, volume 293)


Microdissection techniques have become an important tool to link histomorphology and pathophysiological events using modern methods of molecular biology. They allow isolation of cell clusters or even single cells precisely under optical control from complex tissue structures for further analysis of DNA, RNA, and proteins. In particular, the fragile RNA molecules can be preserved during microdissection so that gene expression and regulation measurement become feasible in a cell type-specific manner within complex tissues. This report focuses on and outlines the procedures for RNA investigation, from tissue fixation, sectioning, and staining to downstream applications (RT-PCR, mRNA quantification, and mRNA preamplification). Standards for the preparation of RNA from frozen and formalin-fixed tissues are presented. Specific protocols are given for both the isolation of RNA from small numbers of cells (50 cells) as well as for larger cell numbers. While most of the procedures are identical for the microdissection systems, special features of each technique are mentioned.

Key Words

Laser microdissection gene expression formalin fixation immunohistochemistry immunofluorescence mRNA quantification real-time PCR RNA preamplification LCM 


  1. 1.
    Kuppers, R., Zhao, M., Hansmann, M. L., and Rajewsky, K. (1993) Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J. 12, 4955–4967.PubMedGoogle Scholar
  2. 2.
    Moskaluk, C. A. and Kern, S. E. (1997) Microdissection and polymerase chain reaction amplification of genomic DNA from histological tissue sections. Am. J. Pathol. 150, 1547–1552.PubMedGoogle Scholar
  3. 3.
    Hiller, T., Snell, L., and Watson, P. H. (1996) Microdissection RT-PCR analysis of gene expression in pathologically defined frozen tissue sections. Biotechniques 21, 38–40, 42, 44.PubMedGoogle Scholar
  4. 4.
    Walch, A., Specht, K., Smida, J., Aubele, M., Zitzelsberger, H., Hofler, H., and Werner, M. (2001) Tissue microdissection techniques in quantitative genome and gene expression analyses. Histochem. Cell Biol. 115, 269–276.PubMedGoogle Scholar
  5. 5.
    Macabeo-Ong, M., Ginzinger, D. G., Dekker, N., McMillan, A., Regezi, J. A., Wong, D. T., and Jordan, R. C. (2002) Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses. Mod. Pathol. 15, 979–987.PubMedCrossRefGoogle Scholar
  6. 6.
    Gillespie, J. W., Best, C. J., Bichsel, V. E., Cole, K. A., Greenhut, S. F., Hewitt, S. M., et al. (2002) Evaluation of non-formalin tissue fixation for molecular profiling. Am. J. Pathol. 160, 449–457.PubMedCrossRefGoogle Scholar
  7. 7.
    Shibutani, M., Uneyama, C., Miyazaki, K., Toyoda, K., and Hirose, M. (2000) Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens. Lab. Invest. 80, 199–208.PubMedCrossRefGoogle Scholar
  8. 8.
    Finkelstein, S. D., Dhir, R., Rabinovitz, M., Bischeglia, M., Swalsky, P. A., De Flavia, P., et al. (1999) Cold-temperature plastic resin embedding of liver for DNA-and RNA-based genotyping. J. Mol. Diagn. 1, 17–22.PubMedCrossRefGoogle Scholar
  9. 9.
    d’Amore, F., Stribley, J. A., Ohno, T., Wu, G., Wickert, R. S., Delabie, J., et al. (1997) Molecular studies on single cells harvested by micromanipulation from archival tissue sections previously stained by immunohistochemistry or nonisotopic in situ hybridization. Lab. Invest. 76, 219–224.PubMedGoogle Scholar
  10. 10.
    Fink, L., Kinfe, T., Seeger, W., Ermert, L., Kummer, W., and Bohle, R.M. (2000) Immunostaining for cell picking and real-time mRNA quantitation. Am. J. Pathol. 157, 1459–1466.PubMedCrossRefGoogle Scholar
  11. 11.
    Gjerdrum, L.M., Lielpetere, I., Rasmussen, L. M., Bendix, K., and Hamilton-Dutoit, S. (2001) Laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis: identification of cell populations using immunohistochemistry and in situ hybridization. J. Mol. Diagn. 3, 105–110.PubMedCrossRefGoogle Scholar
  12. 12.
    Fend, F., Emmert-Buck, M. R., Chuaqui, R., Cole, K., Lee, J., Liotta, L.A., and Raffeld, M. (1999) Immuno-LCM: laser capture microdissection of immuno-stained frozen sections for mRNA analysis. Am. J. Pathol. 154, 61–66.PubMedCrossRefGoogle Scholar
  13. 13.
    Burton, M. P., Schneider, B. G., Brown, R., Escamilla-Ponce, N., and Gulley, M. L. (1998) Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues. Biotechniques 24, 86–92.PubMedGoogle Scholar
  14. 14.
    Murase, T., Inagaki, H., and Eimoto, T. (2000) Influence of histochemical and immunohistochemical stains on polymerase chain reaction. Mod. Pathol. 13, 147–151.PubMedCrossRefGoogle Scholar
  15. 15.
    Fink, L., Kinfe, T., Stein, M.M., Ermert, L., Hanze, J., Kummer, W., et al. (2000) Immunostaining and laser-assisted cell picking for mRNA analysis. Lab. Invest. 80, 327–333.PubMedGoogle Scholar
  16. 16.
    Kohda, Y., Murakami, H., Moe, O. W., and Star, R.A. (2000) Analysis of segmental renal gene expression by laser capture microdissection. Kidney Int. 57, 321–331.PubMedCrossRefGoogle Scholar
  17. 17.
    Murakami, H., Liotta, L., and Star, R.A. (2000) IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis. Kidney Int. 58, 1346–1353.PubMedCrossRefGoogle Scholar
  18. 18.
    Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., Chuaqui, R. F., Zhuang, Z., Goldstein, S. R., et al. (1996) Laser capture microdissection. Science 274, 998–1001.PubMedCrossRefGoogle Scholar
  19. 19.
    Schutze, K., Becker, I., Becker, K. F., Thalhammer, S., Stark, R., Heckl, W. M., et al. (1997) Cut out or poke in-the key to the world of single genes: laser micro-manipulation as a valuable tool on the look-out for the origin of disease. Genet. Anal. 14, 1–8.PubMedGoogle Scholar
  20. 20.
    Schutze, K. and Lahr, G. (1998) Identification of expressed genes by laser-mediated manipulation of single cells. Nat. Biotechnol. 16, 737–742.PubMedCrossRefGoogle Scholar
  21. 21.
    Kolble, K. (2000) The Leica microdissection system: design and applications. J. Mol. Med. 78, B24–B25.PubMedGoogle Scholar
  22. 22.
    Fink, L., Stahl, U., Ermert, L., Kummer, W., Seeger, W., and Bohle, R. M. (1999) Rat porphobilinogen deaminase gene: a pseudogene-free internal standard for laser-assisted cell picking. Biotechniques 26, 510–516.PubMedGoogle Scholar
  23. 23.
    To, M. D., Done, S. J., Redston, M., and Andrulis, I. L. (1998) Analysis of mRNA from microdissected frozen tissue sections without RNA isolation. Am. J. Pathol. 153, 47–51.PubMedCrossRefGoogle Scholar
  24. 24.
    Cordell, J. L., Falini, B., Erber, W. N., Ghosh, A. K., Abdulaziz, Z., MacDonald, S., et al. (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J. Histochem. Cytochem. 32, 219–229.PubMedGoogle Scholar
  25. 25.
    Imamichi, Y., Lahr, G., and Wedlich, D. (2001) Laser-mediated microdissection of paraffin sections from Xenopus embryos allows detection of tissue-specific expressed mRNAs. Dev. Genes Evol. 211, 361–366.PubMedCrossRefGoogle Scholar
  26. 26.
    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.PubMedCrossRefGoogle Scholar
  27. 27.
    Stanta, G. and Schneider, C. (1991) RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification. Biotechniques 11, 304, 306, 308.PubMedGoogle Scholar
  28. 28.
    Goldsworthy, S. M., Stockton, P. S., Trempus, C. S., Foley, J. F., and Maronpot, R. R. (1999) Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol. Carcinog. 25, 86–91.PubMedCrossRefGoogle Scholar
  29. 29.
    Fink, L., Seeger, W., Ermert, L., Hanze, J., Stahl, U., Grimminger, F., et al. (1998) Real-time quantitative RT-PCR after laser-assisted cell picking. Nat. Med. 4, 1329–1333.PubMedCrossRefGoogle Scholar
  30. 30.
    Bohle, R. M., Hartmann, E., Kinfe, T., Ermert, L., Seeger, W., and Fink, L. (2000) Cell type-specific mRNA quantitation in non-neoplastic tissues after laser-assisted cell. Pathobiology 68, 191–195.PubMedCrossRefGoogle Scholar
  31. 31.
    Nagasawa, Y., Takenaka, M., Matsuoka, Y., Imai, E., and Hori, M. (2000) Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting. Kidney Int. 57, 717–723.PubMedCrossRefGoogle Scholar
  32. 32.
    Cohen, C. D., Frach, K., Schlondorff, D., and Kretzler, M. (2002) Quantitative gene expression analysis in renal biopsies: a novel protocol for a high-throughput multicenter application. Kidney Int. 61, 133–140.PubMedCrossRefGoogle Scholar
  33. 33.
    Cohen, C. D., Grone, H. J., Grone, E. F., Nelson, P. J., Schlondorff, D., and Kretzler, M. (2002) Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue. Kidney Int. 61, 125–132.PubMedCrossRefGoogle Scholar
  34. 34.
    El-Sherif, A. M., Seth, R., Tighe, P. J., and Jenkins, D. (2001) Quantitative analysis of IL-10 and IFN-gamma mRNA levels in normal cervix and human papillomavirus type 16 associated cervical precancer. J. Pathol. 195, 179–185.PubMedCrossRefGoogle Scholar
  35. 35.
    Steger, K., Fink, L., Klonisch, T., Bohle, R. M., and Bergmann, M. (2002) Prota-mine-1 and-2 mRNA in round spermatids is associated with RNA-binding proteins. Histochem. Cell Biol. 117, 227–234.PubMedCrossRefGoogle Scholar
  36. 36.
    Luo, L., Salunga, R. C., Guo, H., Bittner, A., Joy, K. C., Galindo, J. E., et al. (1999) Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat. Med. 5, 117–122.PubMedCrossRefGoogle Scholar
  37. 37.
    Ohyama, H., Zhang, X., Kohno, Y., Alevizos, I., Posner, M., Wong, D. T., and Todd, R. (2000) Laser capture microdissection-generated target sample for high-density oligonucleotide array hybridization. Biotechniques 29, 530–536.PubMedGoogle Scholar
  38. 38.
    Luzzi, V., Holtschlag, V., and Watson, M. A. (2001) Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays. Am. J. Pathol. 158, 2005–2010.PubMedCrossRefGoogle Scholar
  39. 39.
    Spirin, K. S., Ljubimov, A. V., Castellon, R., Wiedoeft, O., Marano, M., Sheppard, D., et al. (1999) Analysis of gene expression in human bullous keratopathy corneas containing limiting amounts of RNA. Invest. Ophthalmol. Vis.Sci. 40, 3108–3115.PubMedGoogle Scholar
  40. 40.
    Vernon, S. D., Unger, E. R., Rajeevan, M., Dimulescu, I. M., Nisenbaum, R., and Campbell, C. E. (2000) Reproducibility of alternative probe synthesis approaches for gene expression profiling with arrays. J. Mol. Diagn. 2, 124–127.PubMedCrossRefGoogle Scholar
  41. 41.
    Leethanakul, C., Patel, V., Gillespie, J., Pallente, M., Ensley, J. F., Koontongkaew, S., et al. (2000) Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA. Oncogene 19, 3220–3224.PubMedCrossRefGoogle Scholar
  42. 42.
    Fink, L., Kohlhoff, S., Stein, M. M., Hanze, J., Weissmann, N., Rose, F., et al. (2002) cDNA array hybridization after laser-assisted microdissection from non-neoplastic tissue. Am. J. Pathol. 160, 81–90.PubMedCrossRefGoogle Scholar
  43. 43.
    Chenchik, A., Zhu, Y. Y., Diachenko, L., Li, R., Hill, J., and Siebert, P. D. (1998) Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR, in Gene Cloning and Analysis by RT-PCR, BioTechniques Books, Westborough, MA, pp. 305–319.Google Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Ludger Fink
    • 1
  • Rainer Maria Bohle
    • 2
  1. 1.Department of PathologyUniversity of GiessenGiessenGermany
  2. 2.Department of PathologyJustus-Liebig-UniversityGiessenGermany

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