microRNAs in Cancer Chemoprevention: Method to Isolate Them from Fresh Tissues

  • Federica Ganci
  • Giovanni BlandinoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1379)


microRNAs are 22-nucleotide-long double-strand small RNAs, able to modulate gene expression at posttranscriptional level, degrading mRNA and/or impairing translation. They have been shown to regulate mRNA and protein abundance and to participate in many regulatory circuits controlling developmental timing, cell proliferation and differentiation, apoptosis and stress response. Notably, microRNA activity has been correlated to the pathogenesis of cancer; they are aberrantly expressed in solid and hematological tumors, suggesting that they could function as oncogenes or tumor suppressors. The emerging role of miRNAs in the carcinogenesis and tumor progression has provided opportunities for their clinical application in the capacity of cancer detection, diagnosis, and prognosis prediction. Here, we describe the experimental protocol used to isolate microRNAs from human tissues coming from head and neck, mesothelioma, and thymoma tumors in order to perform microarray and RT-qPCR experiments.

Key words

microRNA Fresh tissues RNA extraction HNSCC Mesothelioma Thymoma 


  1. 1.
    Lagos-Quintana M, Rauhut R, Lendeckel W et al (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858CrossRefPubMedGoogle Scholar
  2. 2.
    Tang G, Tang X, Mendu V et al (2008) The art of microRNA: various strategies leading to gene silencing via an ancient pathway. Biochim Biophys Acta 1779:655–662CrossRefPubMedGoogle Scholar
  3. 3.
    Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355CrossRefPubMedGoogle Scholar
  4. 4.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefPubMedGoogle Scholar
  5. 5.
    Friedman RC, Farh KK, Burge CB et al (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19:92–105PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Landgraf P, Rusu M, Sheridan R et al (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129:1401–1414PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Ross JS, Carlson JA, Brock G (2007) miRNA: the new gene silencer. Am J Clin Pathol 128:830–836CrossRefPubMedGoogle Scholar
  8. 8.
    Visone R, Croce CM (2009) MiRNAs and cancer. Am J Pathol 174:1131–1138PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Lu J, Getz G, Miska EA et al (2005) MicroRNA expression profiles classify human cancers. Nature 435:834–838CrossRefPubMedGoogle Scholar
  10. 10.
    Pritchard CC, Cheng HH, Tewari M (2012) MicroRNA profiling: approaches and considerations. Nat Rev Genet 13:358–369PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    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 7:36PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Cioce M, Ganci F, Canu V et al (2014) Protumorigenic effects of mir-145 loss in malignant pleural mesothelioma. Oncogene 33:5319PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Ganci F, Sacconi A, Bossel Ben-Moshe N et al (2013) Expression of TP53 mutation-associated microRNAs predicts clinical outcome in head and neck squamous cell carcinoma patients. Ann Oncol 24:3082–3088PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Ganci F, Vico C, Korita E et al (2014) MicroRNA expression profiling of thymic epithelial tumors. Lung Cancer 85:197–204CrossRefPubMedGoogle Scholar
  15. 15.
    Mraz M, Malinova K, Mayer J et al (2009) MicroRNA isolation and stability in stored RNA samples. Biochem Biophys Res Commun 390:1–4CrossRefPubMedGoogle Scholar
  16. 16.
    Podolska A, Kaczkowski B, Litman T et al (2011) How the RNA isolation method can affect microRNA microarray results. Acta Biochim Pol 58:535–540PubMedGoogle Scholar
  17. 17.
    Hammerle-Fickinger A, Riedmaier I, Becker C et al (2010) Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses. Biotechnol Lett 32:35–44CrossRefPubMedGoogle Scholar
  18. 18.
    Ibberson D, Benes V, Muckenthaler MU et al (2009) RNA degradation compromises the reliability of microRNA expression profiling. BMC Biotechnol 9:102PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Wang WX, Wilfred BR, Baldwin DA et al (2008) Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue. Biochim Biophys Acta 1779:749–757PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Liu A, Tetzlaff MT, Vanbelle P et al (2009) MicroRNA expression profiling outperforms mRNA expression profiling in formalin-fixed paraffin-embedded tissues. Int J Clin Exp Pathol 2:519–527PubMedCentralPubMedGoogle Scholar
  21. 21.
    Zhang X, Chen J, Radcliffe T et al (2008) An array-based analysis of microRNA expression comparing matched frozen and formalin-fixed paraffin-embedded human tissue samples. J Mol Diagn 10:513–519PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Doleshal M, Magotra AA, Choudhury B et al (2008) Evaluation and validation of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues. J Mol Diagn 10:203–211PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Translational Oncogenomics UnitItalian National Cancer Institute “Regina Elena”RomeItaly
  2. 2.Laboratory of Translational Oncogenomics, Molecular Medicine DepartmentRegina Elena National Cancer InstituteRomeItaly

Personalised recommendations