Skip to main content
SpringerLink
Log in

Laser Microdissection Workflow for Isolating Nucleic Acids from Fixed and Frozen Tissue Samples

  • Protocol
  • First Online:
Book cover Laser Capture Microdissection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1723))

Abstract

Laser Capture Microdissection has earned a permanent place among modern techniques connecting histology and molecular biology. Laser Capture Microdissection has become an invaluable tool in medical research as a means for collection of specific cell populations isolated from their environment. Such genomic sample enrichment dramatically increases the sensitivity and precision of downstream molecular assays used for biomarker discovery, monitoring disease onset and progression, and in the development of personalized medicine. The diversity of research targets (cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, frozen tissues, archival repository slides, etc.) and scientific objectives present a challenge in establishing standard protocols for Laser Capture Microdissection. In the present chapter, we share our experiences in design and successful execution of numerous diverse microdissection projects, and provide considerations to be taken into account in planning a microdissection study. Our workflow and protocols are standardized for a wide range of animal and human tissues and adapted to downstream analysis platforms.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Emmert-Buck MR, Bonner RF, Smith P et al (1996) Laser capture microdissection. Science 274:998–1001

    Article  CAS  Google Scholar 

  2. Bonner RF, Emmert-Buck MR, Cole K et al (1997) Laser capture microdissection: molecular analysis of tissue. Science 278:1481–1483

    Article  CAS  Google Scholar 

  3. Murray GI (2007) Overview of laser microdissection technologies. Acta Histochem 109:171–176

    Article  Google Scholar 

  4. Domazet B, MacLennan G, Lopez-Beltran A et al (2008) Laser capture microdissection in the genomic and proteomic era: targeting the genetic basis of cancer. Int J Clin Exp Pathol 1:475–488

    CAS  Google Scholar 

  5. Ma XJ, Dahiya S, Richardson EA et al (2009) Gene expression profiling of tumor microenvironment during breast cancer progression. Breast Cancer Res 11:R7

    Article  Google Scholar 

  6. Chimge N, Ruddle F, Bayarsaihan D (2007) Laser-assisted microdissection (LAM) in developmental biology. J Exp Zool B Mol Dev Evol 308:113–118

    Article  Google Scholar 

  7. Jacquet R, Hillyer J, Landis W (2005) Analysis of connective tissues by laser capture microdissection and reverse transcriptase-polymerase chain reaction. Anal Biochem 337:22–34

    Article  CAS  Google Scholar 

  8. Castro NP, Fedorova-Abrams ND, Merchant AS et al (2015) Cripto-1 as a novel therapeutic target for triple negative breast cancer. Oncotarget 6:11910–11929

    Article  Google Scholar 

  9. Gillespie JW, Ahram M, Best CJ et al (2001) The role of tissue microdissection in cancer research. Cancer J 7:32–39

    CAS  Google Scholar 

  10. Baldelli E, Haura EB, Crino L et al (2015) Impact of upfront cellular enrichment by laser capture microdissection on protein and phosphoprotein drug target signaling activation measurements in human lung cancer: implications for personalized medicine. Proteomics Clin Appl 9(9-10):928–937

    Article  CAS  Google Scholar 

  11. Melis M, Diaz G, Kleiner DE et al (2014) Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus-associated hepatocellular carcinoma. J Transl Med 12:230

    Article  Google Scholar 

  12. Cai J, Li T, Huang B et al (2014) The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples. PLoS One 9(4):e95974

    Article  Google Scholar 

  13. Wulfkuhle JD, Speer R, Pierobon M et al (2008) Multiplexed cell signaling analysis of human breast cancer applications for personalized therapy. J Proteome Res 7:1508–1517

    Article  CAS  Google Scholar 

  14. Erickson HS, Albert PS, Gillespie JW et al (2009) Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples. Nat Protoc 4:902–922

    Article  CAS  Google Scholar 

  15. Castro NP, Merchant AS, Sayler K et al (2016) Adaptation of laser microdissection technique for the study of a spontaneous metastatic mammary carcinoma mouse model by NanoString Technologies. PLoS ONE 11(4):e0153270

    Article  Google Scholar 

  16. Keaysa K, Owensa G, Ritchiea A et al (2005) Laser capture microdissection and single-cell RT-PCR without RNA purification. J Immunol Methods 302:90–98

    Article  Google Scholar 

  17. Ross J (1995) mRNA stability in mammalian cells. Microbiol Rev 59:423–450

    CAS  Google Scholar 

  18. Golubeva Y, Rogers K (2009) Collection and preparation of rodent tissue samples for histopathological and molecular studies in carcinogenesis. Methods Mol Biol 511:3–60

    Article  CAS  Google Scholar 

  19. Huang LE, Luzzi V, Ehrig T et al (2002) Optimized tissue processing and staining for laser capture microdissection and nucleic acid retrieval. Methods Enzymol 356:49–62

    Article  CAS  Google Scholar 

  20. Espina V, Wulfkuhle JD, Calvert VS et al (2006) Laser-capture microdissection. Nat Protoc 1:586–603

    Article  CAS  Google Scholar 

  21. Golubeva Y, Smith R, Sternberg R (2013) Optimizing frozen sample preparation for laser microdissection: assessment of CryoJane Tape-Trasfer System®. PLoS One 8(6):e66854

    Article  CAS  Google Scholar 

  22. Gjerdrum LM, Lielpetere I, Rasmussen L et al (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

    Article  CAS  Google Scholar 

  23. Lin DW, Coleman IM, Hawley S, Huang CY, Dumpit R et al (2006) Influence of surgical manipulation on prostate gene expression: implications for molecular correlates of treatment effects and disease prognosis. J Clin Oncol 24:3763–3770

    Article  CAS  Google Scholar 

  24. Chen J, Feigenbaum L, Awasthi P et al (2013) Diabetes induced beta cell expression of IL-15 and IL-15Rα. Proc Natl Acad Sci U S A 110:13534–13539

    Article  CAS  Google Scholar 

  25. Srinivasan M, Sedmak D, Jewell S (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161:1961–1971

    Article  CAS  Google Scholar 

  26. Espina V, Mueller C, Liotta L (2011) Phosphoprotein stability in clinical tissue and its relevance for reverse phase protein microarray technology. Methods Mol Biol 758:23–43

    Article  Google Scholar 

  27. Institute of Laboratory Animal Resources CoLS, National Research Council (1996) Guide for the care and use of laboratory animals. National Academy Press, Washington, DC. 125p

    Google Scholar 

  28. Mueller C, Edmiston K, Carpenter C et al (2011) One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens. PLoS One 6(8):e23780

    Article  CAS  Google Scholar 

  29. Gündisch S, Schott C, Wolff C et al (2013) The PAXgene Tissue System preserves phosphoproteins in human tissue specimens and enables comprehensive protein biomarker research. PLoS One 8(3):e60638

    Article  Google Scholar 

  30. Imbeaud S, Graudens E, Boulanger V et al (2005) Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res 33(6):e56

    Article  Google Scholar 

  31. Pfaffl M, Fleige S, Riedmaier I (2008) Validation of lab-on-chip capillary electrophoresis systems for total RNA quality and quantity control. Biotechnol Biotechnol Eq 22:3

    Article  Google Scholar 

  32. Burton M, Schneider B, Brown R et al (1998) Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues. Biotechniques 24:86–92

    CAS  Google Scholar 

  33. Murase T, Inagaki H, Eimoto T (2000) Influence of histochemical and immunohistochemical stains on polymerase chain reaction. Mod Pathol 13:147–151

    Article  CAS  Google Scholar 

  34. Golubeva Y, Salcedo R, Mueller C et al (2013) Laser capture microdissection for protein and NanoString RNA analysis. Methods Mol Biol 931:213–257

    Article  CAS  Google Scholar 

  35. Okuducu A, Jansen V, Hahne J et al (2003) Influence of histochemical stains on quantitative gene expression analysis after laser-assisted microdissection. Int J Mol Med 11:449–453

    CAS  Google Scholar 

  36. Mojsilovic-Petrovic J, Nesic M, Pen A et al (2004) Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses. J Neurosci Methods 133:39–48

    Article  CAS  Google Scholar 

  37. McLean MH, Hanson M, Gold B et al (2014) Intra-luminal interleukin (il)-27 is a potential future therapeutic for inflammatory bowel disease. Gut 63:A84

    Article  Google Scholar 

  38. Miller R, Winrow C, Spellman D et al (2014) Quantitative proteomics in laser capture microdissected sleep nuclei from rat brain. J Neurogenet 28:136–145

    Article  CAS  Google Scholar 

  39. Fargnoli MC, Pike K, Pfeiffer RM et al (2008) MC1R variants increase risk of melanomas harboring BRAF. J Invest Dermatol 128:2485–2490

    Article  CAS  Google Scholar 

  40. Gundem G, Van Loo P, Kremeyer B et al (2015) The evolutionary history of lethal metastatic prostate cancer. Nature 520:353–357

    Article  CAS  Google Scholar 

  41. Hasumia Y, Babaa M, Ajimab R et al (2009) Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2. Proc Natl Acad Sci U S A 106:18722–18727

    Article  Google Scholar 

  42. Van Bemmel D, Lenz P, Liao L et al (2012) Correlation of LINE-1 methylation levels in patient-matched buffy coat, serum, buccal cell, and bladder tumor tissue DNA samples. Cancer Epidemiol Biomarkers Prev 21:1143–1148

    Article  Google Scholar 

  43. Shi J, Marconett CN, Duan J et al (2014) Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue. Nat Commun 27:3365

    Google Scholar 

  44. Szabova L, Yin C, Bupp S et al (2012) Perturbation of Rb, p53 and Brca1 or Brca2 cooperate in inducing metastatic serous epithelial ovarian cancer. Cancer Res 72:4141–4153

    Article  CAS  Google Scholar 

  45. Xu C, Houck J, Fan W et al (2008) Simultaneous isolation of DNA and RNA from the same cell population obtained by laser capture microdissection for genome and transcriptome profiling. J Mol Diagn 10:129–134

    Article  CAS  Google Scholar 

  46. Fend F, Emmert-Buck M, Chuaqui R et al (1999) Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol 154:61–66

    Article  CAS  Google Scholar 

  47. Tangrea M, Mukherjee S, Gao B et al (2011) Effect of immunohistochemistry on molecular analysis of tissue samples: implications for microdissection technologies. J Histochem Cytochem 59:591

    Article  CAS  Google Scholar 

  48. Demarest T, Murugesan N, Bandana S et al (2012) Rapid expression profiling of brain microvascular endothelial cells by immune-laser capture microdissection coupled to TaqMan Low Density Array. Neurosci Methods 206:200–207

    Article  Google Scholar 

  49. Xiang C, Mezey E, Chen M et al (2004) Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling. Nucleic Acids Res 32:e185

    Article  Google Scholar 

  50. Hann B, Balmain A (2001) Building ‘validated’ mouse models of human cancer. Curr Opin Cell Biol 13:778–784

    Article  CAS  Google Scholar 

  51. Atkinson BG, Walden BD (eds) (1985) Changes in eukaryotic gene expression in response to environmental stress. Academic, Orlando, FL. 379p

    Google Scholar 

  52. Mori N, Mizuno D, Goto S (1978) Increase in ratio of 18S RNA to 28S RNA in the cytoplasm of mouse tissues during aging. Mech Ageing Dev 8:285–297

    Article  CAS  Google Scholar 

  53. Imbeaud S, Auffray C (2005) ‘The 39 steps’ in gene expression profiling: critical issues and proposed best practices for microarray experiments. Drug Discov Today 10:1175–1182

    Article  CAS  Google Scholar 

  54. Botling J, Edlund K, Segersten U et al (2009) Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue. Diagn Mol Pathol 18:44–52

    Article  CAS  Google Scholar 

  55. Kabnick KS, Housman DE (1998) Determinants that contribute to cytoplasmic stability of human c-fos and beta-globin mRNAs are located at several sites in each RNA. Mol Cell Biol 8:8–13

    Google Scholar 

  56. Coudry R, Meireles S, Stoyanova R et al (2007) Successful application of microarray technology to microdissected formalin-fixed, paraffin-embedded tissue. J Mol Diagn 9:70–79

    Article  CAS  Google Scholar 

  57. Danforth D, Warner A, Wangsa D et al (2015) An improved breast epithelial sampling method for molecular profiling and biomarker analysis in women at risk for breast cancer. Breast Cancer (Auckl) 9:31–40

    CAS  Google Scholar 

Download references

Acknowledgments

This Research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Frederick National Laboratory is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 2011; National Academies Press; Washington, D.C.).

Author information

Authors and Affiliations

  1. Cancer Genomic Research Laboratory (CGR), Division of Cancer Epidemiology and Genetics, NCI, FNLCR, Leidos Biomedical Research, Inc, Gaithersburg, MD, USA

    Yelena G. Golubeva

  2. Pathology-Histotechnology Laboratory (PHL), Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    Andrew C. Warner

Authors
  1. Yelena G. Golubeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew C. Warner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yelena G. Golubeva .

Editor information

Editors and Affiliations

  1. School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, United Kingdom

    Graeme I. Murray

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Golubeva, Y.G., Warner, A.C. (2018). Laser Microdissection Workflow for Isolating Nucleic Acids from Fixed and Frozen Tissue Samples. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 1723. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7558-7_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7558-7_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7557-0

  • Online ISBN: 978-1-4939-7558-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation