Gene Expression Profiling in Laser-Microdissected Bone Marrow Megakaryocytes

  • Kais HusseinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 755)


Gene expression analysis of the megakaryocytic lineage requires isolation of megakaryocytes from their bone marrow microenvironment. Laser microdissection of megakaryocytes from diagnostic bone marrow biopsies allows analysis of standardised formalin-fixed samples that reflect the in situ grown status quo of a physiological or pathological condition. Taking into account that in neoplastic proliferation, e.g. myeloproliferative neoplasms, non-neoplastic haematopoietic clones proliferate in parallel, this morphology-based isolation enables selective analysis of the aberrant megakaryocytic population. Two different laser microdissection devices are presented, and the details of RNA extraction and sub-sequent real-time qPCR gene expression analysis of mRNA and microRNA are provided.

Key words

Laser microdissection Bone marrow Megakaryocyte Gene expression Real-time qPCR RNA microRNA 



The author would like to thank his mentors Professor Dr. med. Hans H. Kreipe and Professor Dr. med. Oliver Bock (Institute of Pathology, Hannover Medical School) as well as Dr. phil. Khadra Hussein, Dr. med. Ulrich Thorns and Sir Jan Off.


  1. 1.
    Emmert-Buck MR, Bonner RF, Smith PD et al (1996) Laser capture microdissection. Science 274:998–1001CrossRefGoogle Scholar
  2. 2.
    Lehmann U, Glockner S, Kleeberger W et al (2000) Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction. Am J Pathol 156:1855–1864CrossRefGoogle Scholar
  3. 3.
    Bauer K, Taub S, Parsi K (2004) Ethical issues in tissue banking for research: a brief review of existing organizational policies. Theor Med Bioeth 25:113–142CrossRefGoogle Scholar
  4. 4.
    Caulfield T (2004) Tissue banking, patient rights, and confidentiality: tensions in law and policy. Med Law 23:39–49Google Scholar
  5. 5.
    Bluteau D, Lordier L, Di Stefano et al (2009) Regulation of megakaryocyte maturation and platelet formation. J Thromb Haemost 7:227–234CrossRefGoogle Scholar
  6. 6.
    Birschmann I, Mietner S, Dittrich M et al (2008) Use of functional highly purified human platelets for the identification of new proteins of the IPP signaling pathway. Thromb Res 122:59–68CrossRefGoogle Scholar
  7. 7.
    Swerdlow SH, Campo C, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman, JW (2008) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC, LyonGoogle Scholar
  8. 8.
    Bock O, Hussein K, Kreipe H (2007) Stem cell defects in Philadelphia chromosome negative chronic myeloproliferative disorders: a phenotypic and molecular puzzle? Curr Stem Cell Res Ther 2:253–263CrossRefGoogle Scholar
  9. 9.
    Bock O, Kreipe H, Lehmann U (2001) One-step extraction of RNA from archival biopsies. Anal Biochem 295:116–117CrossRefGoogle Scholar
  10. 10.
    Li J, Smyth P, Flavin R et al (2007) Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells. BMC Biotechnol 7:36CrossRefGoogle Scholar
  11. 11.
    Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefGoogle Scholar
  12. 12.
    Hussein K, Bock O, Theophile K et al (2009) Biclonal expansion and heterogeneous lineage involvement in a case of chronic myeloproliferative disease with concurrent MPLW515L/-JAK2V617F mutation. Blood 113:1391–1392CrossRefGoogle Scholar
  13. 13.
    Hussein K, Theophile K, Buhr T et al (2009) Different lineage involvement in myelodysplastic/myeloproliferative disease with combined MPL and JAK2 mutation. Br J Haematol 146:510–520CrossRefGoogle Scholar
  14. 14.
    Bock O, Schlué J, Lehmann U et al (2002) Megakaryocytes from myeloproliferative disorders show enhanced nuclear bFGF expression. Blood 100:2274–2275CrossRefGoogle Scholar
  15. 15.
    Theophile K, Jonigk D, Kreipe H et al (2008) Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR. Diagn Mol Pathol 17:101–106CrossRefGoogle Scholar
  16. 16.
    Theophile K, Hussein K, Kreipe H et al (2008) Expression profiling of apoptosis-related genes in megakaryocytes: BNIP3 is downregulated in primary myelofibrosis. Exp Hematol 36:1728–1738CrossRefGoogle Scholar
  17. 17.
    Hussein K, Dralle W, Theophile K et al (2009) Megakaryocytic expression of miRNA 10a, 17–5p, 20a and 126 in Philadelphia chromosome-negative myeloproliferative neoplasm. Ann Hematol 88:325–332CrossRefGoogle Scholar
  18. 18.
    Hussein K, Theophile K, Dralle W et al (2009) MicroRNA expression profiling of megakaryocytes in primary myelofibrosis and essential thrombocythemia. Platelets 20:391–400CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Institute of PathologyHannover Medical SchoolHannoverGermany

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