Optimal Molecular Profiling of Tissue and Tissue Components

Defining the Best Processing and Microdissection Methods for Biomedical Applications
  • G. Steven Bova
  • Isam A. Eltoum
  • John A. Kiernan
  • Gene P. Siegal
  • Andra R. Frost
  • Carolyn J. M. Best
  • John W. Gillespie
  • Michael R. Emmert-Buck
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 103)


Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.

Key Words

Molecular profiling tissue processing tissue staining sample processing laser microdissection RNA DNA quality control workflow management 


  1. 1.
    Eltoum, I. A., Siegal, G. P., and Frost, A. R. (2002) Microdissection of histologic sections: Past, present, and future. Adv. Anat. Pathol. 9, 316–322.PubMedCrossRefGoogle Scholar
  2. 2.
    Srinivasan, M., Sedmak, D., and Jewell, S. (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol. 161, 1961–1971.PubMedCrossRefGoogle Scholar
  3. 3.
    Best, C. J. and Emmert-Buck, M. R. (2001) Molecular profiling of tissue samples using laser capture microdissection. Expert. Rev. Mol. Diagn. 1, 53–60.PubMedCrossRefGoogle Scholar
  4. 4.
    Ahram, M., Flaig, M. J., Gillespie, J. W., et al. (2003) Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications. Proteomics 3, 413–421.PubMedCrossRefGoogle Scholar
  5. 5.
    Englert, C. R., Baibakov, G. V., and Emmert-Buck, M. R. (2000) Layered expression scanning: Rapid molecular profiling of tumor samples. Cancer Res. 60, 1526–1530.PubMedGoogle Scholar
  6. 6.
    Fend, F., Emmert-Buck, M. R., Chuaqui, R., et al. (1999) Immuno-LCM: Laser capture microdissection of immunostained frozen sections for mRNA analysis. Am. J. Pathol. 154, 61–66.PubMedCrossRefGoogle Scholar
  7. 7.
    Gillespie, J. W., Best, C. J., Bichsel, V. E., et al. (2002) Evaluation of non-formalin tissue fixation for molecular profiling studies. Am. J. Pathol. 160, 449–457.PubMedCrossRefGoogle Scholar
  8. 8.
    Gillespie, J. W., Ahram, M., Best, C. J., et al. (2001) The role of tissue microdissection in cancer research. Cancer J. 7, 32–39.PubMedGoogle Scholar
  9. 9.
    Ornstein, D. K., Gillespie, J. W., Paweletz, C. P., et al. (2000) Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines. Electrophoresis 21, 2235–2242.PubMedCrossRefGoogle Scholar
  10. 10.
    Simone, N. L., Remaley, A. T., Charboneau, L., et al. (2000) Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection. Am. J. Pathol. 156, 445–452.PubMedCrossRefGoogle Scholar
  11. 11.
    Bancroft, J. D. and Gamble, M. (2002) Theory and Practice of Histological Techniques, 5th ed. Edinburgh: Churchill Livingstone.Google Scholar
  12. 12.
    Carson, F. L. (2003) Histotechnology: A Self Instructional Text, 2nd ed. Chicago: ASCP Press.Google Scholar
  13. 13.
    Kiernan, J. A. (2001) Histological and Histochemical Methods, 3rd ed. Oxford: Oxford University Press.Google Scholar
  14. 14.
    Kiernan, J. A. and Mason, I. (eds.) (2002) Microscopy and Histology for Molecular Biologists: A User’s Guide. London: Portland Press.Google Scholar
  15. 15.
    Histonet Listserver Information
  16. 16.
    Gassmann, M. (2003) Quality Assurance of RNA derived from laser microdissected tissue samples obtained by the PALM(R) MicroBeam System using the RNA 6000 Pico LabChip(R) kit. 1-8. 2003. Agilent Technologies.Google Scholar
  17. 17.
    Horobin, R. W. and Kiernan, J. A. (eds.) (2002) Conn’s Biological Stains: A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine, 10th ed. Published for the Biological Stain Commission by BIOS Scientific Publishers, Distributed in US by Springer-Verlag (U.S.), Oxford, UK.Google Scholar
  18. 18.
    Zhuang, Z., Bertheau, P., Emmert-Buck, M. R., et al. (1995) A microdissection technique for archival DNA analysis of specific cell populations in lesions <1 mm in size. Am. J. Pathol. 146, 620–625.PubMedGoogle Scholar
  19. 19.
    Lee, J. Y., Dong, S. M., Kim, S. Y., Yoo, N. J., Lee, S. H., and Park, W. S. (1998) A simple, precise and economical microdissection technique for analysis of genomic DNA from archival tissue sections. Virchows Arch. 433, 305–309.PubMedCrossRefGoogle Scholar
  20. 20.
    Gupta, S. K, Douglas-Jones, A. G., and Morgan, J. M. (1997) Microdissection of stained archival tissue. Mol. Pathol. 50, 218–220.PubMedCrossRefGoogle Scholar
  21. 21.
    Harsch, M., Bendrat, K., Hofmeier, G., Branscheid, D., and Niendorf, A. (2001) A new method for histological microdissection utilizing an ultrasonically oscillating needle: Demonstrated by differential mRNA expression in human lung carcinoma tissue. Am. J. Pathol. 158, 1985–1990.PubMedCrossRefGoogle Scholar
  22. 22.
    Beltinger, C. P. and Debatin, K. M. (1998) A simple combined microdissection and aspiration device for the rapid procurement of single cells from clinical peripheral blood smears. Mol. Pathol. 51, 233–236.PubMedCrossRefGoogle Scholar
  23. 23.
    Zhang, Z., Nakamura, M., Taniguchi, E., Shan, L., Yokoi, T., and Kakudo, K. (1997) A simple approach to single-cell microdissection and molecular analysis. Anal. Quant. Cytol. Histol. 19, 514–518.PubMedGoogle Scholar
  24. 24.
    Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., et al. (1996) Laser capture microdissection [see Comments]. Science 274, 998–1001.PubMedCrossRefGoogle Scholar
  25. 25.
    Curran, S., McKay, J. A., McLeod, H. L., and Murray, G. I. (2000) Laser capture microscopy. Mol. Pathol. 53, 64–68.PubMedCrossRefGoogle Scholar
  26. 26.
    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
  27. 27.
    Lawrie, L. C, Curran, S., McLeod, H. L., Fothergill, J. E., and Murray, G. I. (2001) Application of laser capture microdissection and proteomics in colon cancer. Mol. Pathol. 54, 253–258.PubMedCrossRefGoogle Scholar
  28. 28.
    Craven, R. A. and Banks, R. E. (2002) Use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis. Methods Enzymol. 356, 33–49.PubMedCrossRefGoogle Scholar
  29. 29.
    Nakazono, M., Qiu, F., Borsuk, L. A., and Schnable, P. S. (2003) Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: Identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15, 583–596.PubMedCrossRefGoogle Scholar
  30. 30.
    Luzzi, V., Mahadevappa, M., Raja, R., Warrington, J. A., and Watson, M. A. (2003) Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis. J. Mol. Diagn. 5, 9–14.PubMedCrossRefGoogle Scholar
  31. 31.
    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.PubMedCrossRefGoogle Scholar
  32. 32.
    Fink, L., Kohlhoff, S., Stein, M. M., Hanze, J., et al. (2002) cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue. Am. J. Pathol. 160, 81–90.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.
    Kleeberger, W., Rothamel, T., Glockner, S., Lehmann, U., and Kreipe, H. (2000) Laser-assisted microdissection and short tandem repeat PCR for the investigation of graft chimerism after solid organ transplantation. Pathobiology 68, 196–201.PubMedCrossRefGoogle Scholar
  35. 35.
    Inoue, K., Sakurada, Y., Murakami, M., Shirota, M., and Shirota, K. (2003) Detection of gene expression of vascular endothelial growth factor and flk-1 in the renal glomeruli of the normal rat kidney using the laser microdissection system. Virchows Arch. 442, 159–162.PubMedGoogle Scholar
  36. 36.
    Mori, M., Mimori, K., Yoshikawa, Y., et al. (2002) Analysis of the gene-expression profile regarding the progression of human gastric carcinoma. Surgery 131, S39–S47.PubMedCrossRefGoogle Scholar
  37. 37.
    Brockhoff, G., Fleischmann, S., Meier, A., Wachs, F. P., Hofstaedter, F., and Knuechel, R. (1999) Use of a mechanical dissociation device to improve standardization of flow cytometric cytokeratin DNA measurements of colon carcinomas. Cytometry 38, 184–191.PubMedCrossRefGoogle Scholar
  38. 38.
    Pertoft, H. (2000) Fractionation of cells and subcellular particles with Percoll. J. Biochem. Biophys. Methods 44, 1–30.PubMedCrossRefGoogle Scholar
  39. 39.
    Tai, Y. T., Teoh, G, Shima, Y., et al. (2000) Isolation and characterization of human multiple myeloma cell enriched populations. J. Immunol. Methods 235, 11–19.PubMedCrossRefGoogle Scholar
  40. 40.
    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
  41. 41.
    Goelz, S. E., Hamilton, S. R., and Vogelstein, B. (1985) Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem. Biophys. Res. Commun. 130, 118–126.PubMedCrossRefGoogle Scholar
  42. 42.
    Emmert-Buck, M. R., Gillespie, J. W., Paweletz, C. P., et al. (2000) An approach to proteomic analysis of human tumors. Mol. Carcinog. 27, 158–165.PubMedCrossRefGoogle Scholar
  43. 43.
    Ikeda, K., Monden, T., Kanoh, T., et al. (1998) Extraction and analysis of diag-nostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J. Histochem. Cytochem. 46, 397–403.PubMedGoogle Scholar
  44. 44.
    Novelli, M., Savoia, P., Cambieri, I., et al. (2000) Collagenase digestion and mechanical disaggregation as a method to extract and immunophenotype tumour lymphocytes in cutaneous T-cell lymphomas. Clin. Exp. Dermatol. 25, 423–431.PubMedCrossRefGoogle Scholar
  45. 45.
    Maitra, A., Wistuba, I. I., Virmani, A. K., et al. (1999) Enrichment of epithelial cells for molecular studies. Nat. Med. 5, 459–463.PubMedCrossRefGoogle Scholar
  46. 46.
    Guerrero, R. B., Batts, K. P., Brandhagen, D. J., Germer, J. J., Perez, R. G., and Persing, D. H. (1997) Effects of formalin fixation and prolonged block storage on detection of hepatitis C virus RNA in liver tissue. Diagn. Mol. Pathol. 6, 277–281.PubMedCrossRefGoogle Scholar
  47. 47.
    Ohyama, H., Zhang, X., Kohno, Y., and Alevizos, M. (2000) Laser capture micro-dissection-generated target sample for high-density oligonucleotide array hybridization. Biotechniques 29, 530–536.PubMedGoogle Scholar
  48. 48.
    Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.PubMedCrossRefGoogle Scholar
  49. 49.
    Maunsbach, A. B. (1966) The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. II. Effects of varying osmolality, ionic strength, buffer system and fixative concentration of glutaraldehyde solutions. J. Ultrastruct. Res. 15, 283–309.PubMedCrossRefGoogle Scholar
  50. 50.
    Morales, A. R., Essenfeld, H., Essenfeld, E., Duboue, M. C, Vincek, V., and Nadji, M. (2002) Continuous-specimen-flow, high-throughput, 1-hour tissue processing. A system for rapid diagnostic tissue preparation. Arch. Pathol. Lab. Med. 126, 583–590.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • G. Steven Bova
    • 1
  • Isam A. Eltoum
    • 2
    • 3
    • 4
    • 5
  • John A. Kiernan
    • 6
  • Gene P. Siegal
    • 2
    • 3
    • 4
    • 5
  • Andra R. Frost
    • 2
    • 3
    • 4
    • 5
  • Carolyn J. M. Best
    • 7
  • John W. Gillespie
    • 8
  • Michael R. Emmert-Buck
    • 7
  1. 1.Departments of P of Genetic MedicineThe Johns Hopkins Medical InstitutionsBaltimore
  2. 2.Department of PathologyUniversity of Alabama at BirminghamBirmingham
  3. 3.Department of Cell BiologyUniversity of Alabama at BirminghamBirmingham
  4. 4.Department of SurgeryUniversity of Alabama at BirminghamBirmingham
  5. 5.the UAB Comprehensive Cancer CenterUniversity of Alabama at BirminghamBirmingham
  6. 6.Department of Anatomy and Cell BiologyUniversity of Western OntarioLondonCanada
  7. 7.Pathogenetics Unit, National Cancer InstituteNational Institutes of HealthBethesda
  8. 8.Science Applications International CorporationNational Cancer InstituteBethesda

Personalised recommendations