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Fully Automated Fluorescence-Based Four-Color Multiplex Assay for Co-detection of MicroRNA and Protein Biomarkers in Clinical Tissue Specimens

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In Situ Hybridization Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1211))

Abstract

The application of locked nucleic acid chemistry for microRNA detection by in situ hybridization, and thereby visualization of microRNA expression at single-cell resolution, has contributed to our understanding of the roles that these short noncoding regulatory RNAs play during development, physiology, and disease. Several groups have implemented chromogenic-based and fluorescence-based protocols to detect microRNA expression in formalin-fixed paraffin-embedded clinical tissue specimens. These emerging robust and reproducible tissue slide-based assays are valid tools to bring about the clinical application of in situ microRNA detection for routine diagnostics. Here, I describe a fully automated fluorescence-based four-color multiplex assay for co-detection of a microRNA (e.g., let-7a, miR-10b, miR-21, miR-34a, miR-126, miR-145, miR-155, miR-205, miR-210), reference RNA (e.g., U6 snRNA, 18S rRNA), and protein markers (e.g., CD11b, CD20, CD45, collagen I, cytokeratin 7, cytokeratin 19, smooth muscle actin, tubulin, vimentin) in FDA-approved Leica Bond-MAX staining station.

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References

  1. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864

    Article  PubMed  CAS  Google Scholar 

  2. Lau NC, Lim LP, Weinstein EG et al (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862

    Article  PubMed  CAS  Google Scholar 

  3. Lagos-Quintana M, Rauhut R, Lendeckel W et al (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858

    Article  PubMed  CAS  Google Scholar 

  4. Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355

    Article  PubMed  CAS  Google Scholar 

  5. Mendell JT, Olson EN (2012) MicroRNAs in stress signaling and human disease. Cell 148:1172–1187

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Krol J, Loedige I, Filipowicz W (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11:597–610

    PubMed  CAS  Google Scholar 

  7. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  8. Ketting RF (2011) MicroRNA biogenesis and function: an overview. Adv Exp Med Biol 700:1–14

    Article  PubMed  Google Scholar 

  9. Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. Garzon R, Fabbri M, Cimmino A et al (2006) MicroRNA expression and function in cancer. Trends Mol Med 12:580–587

    Article  PubMed  CAS  Google Scholar 

  11. Plasterk RH (2006) Micro RNAs in animal development. Cell 124:877–881

    Article  PubMed  CAS  Google Scholar 

  12. Kloosterman WP, Plasterk RH (2006) The diverse functions of microRNAs in animal development and disease. Dev Cell 11:441–450

    Article  PubMed  CAS  Google Scholar 

  13. Curtis C, Shah SP, Chin SF et al (2012) The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486:346–352

    PubMed  CAS  PubMed Central  Google Scholar 

  14. Yuan Y, Failmezger H, Rueda OM et al (2012) Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling. Sci Transl Med 4:157ra143

    Article  PubMed  Google Scholar 

  15. Martinez P, Birkbak NJ, Gerlinger M et al (2013) Parallel evolution of tumour subclones mimics diversity between tumours. J Pathol 230:356–364

    Article  PubMed  CAS  Google Scholar 

  16. Gerlinger M, Rowan AJ, Horswell S et al (2012) Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366:883–892

    Article  PubMed  CAS  Google Scholar 

  17. Heppner GH (1984) Tumor heterogeneity. Cancer Res 44:2259–2265

    PubMed  CAS  Google Scholar 

  18. Klein CA (2013) Selection and adaptation during metastatic cancer progression. Nature 501:365–372

    Article  PubMed  CAS  Google Scholar 

  19. Bedard PL, Hansen AR, Ratain MJ et al (2013) Tumour heterogeneity in the clinic. Nature 501:355–364

    Article  PubMed  CAS  Google Scholar 

  20. Junttila MR, de Sauvage FJ (2013) Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501:346–354

    Article  PubMed  CAS  Google Scholar 

  21. Burrell RA, McGranahan N, Bartek J et al (2013) The causes and consequences of genetic heterogeneity in cancer evolution. Nature 501:338–345

    Article  PubMed  CAS  Google Scholar 

  22. Meacham CE, Morrison SJ (2013) Tumour heterogeneity and cancer cell plasticity. Nature 501:328–337

    Article  PubMed  CAS  Google Scholar 

  23. Marte B (2013) Tumour heterogeneity. Nature 501:327

    Article  PubMed  CAS  Google Scholar 

  24. Almendro V, Marusyk A, Polyak K (2013) Cellular heterogeneity and molecular evolution in cancer. Annu Rev Pathol 8:277–302

    Article  PubMed  CAS  Google Scholar 

  25. Marusyk A, Almendro V, Polyak K (2012) Intra-tumour heterogeneity: a looking glass for cancer? Nat Rev Cancer 12:323–334

    Article  PubMed  CAS  Google Scholar 

  26. Wood LD, Parsons DW, Jones S et al (2007) The genomic landscapes of human breast and colorectal cancers. Science 318:1108–1113

    Article  PubMed  CAS  Google Scholar 

  27. Jones S, Zhang X, Parsons DW et al (2008) Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321:1801–1806

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  28. Nelson PT, Wilfred BR (2009) In situ hybridization is a necessary experimental complement to microRNA (miRNA) expression profiling in the human brain. Neurosci Lett 466:69–72

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Sempere LF (2011) Integrating contextual miRNA and protein signatures for diagnostic and treatment decisions in cancer. Expert Rev Mol Diagn 11:813–827

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Sempere LF, Kauppinen S (2009) Translational implications of microRNAs in clinical diagnostics and therapeutics. In: Bradshaw RA, Dennis EA (eds) Handbook of cell signaling, 2nd edn. Academic, Oxford, pp 2965–2981

    Google Scholar 

  31. Zhang X, Lu X, Lopez-Berestein G et al (2013) In situ hybridization-based detection of microRNAs in human diseases. MicroRNA Diagn Ther 1:12–23

    Google Scholar 

  32. Nana-Sinkam SP, Croce CM (2013) Clinical applications for microRNAs in cancer. Clin Pharmacol Ther 93:98–104

    Article  PubMed  CAS  Google Scholar 

  33. Ventura A, Jacks T (2009) MicroRNAs and cancer: short RNAs go a long way. Cell 136:586–591

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  34. Kloosterman WP, Wienholds E, de BE et al (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3:27–29

    Article  PubMed  CAS  Google Scholar 

  35. Wienholds E, Kloosterman WP, Miska E et al (2005) MicroRNA expression in zebrafish embryonic development. Science 309:310–311

    Article  PubMed  CAS  Google Scholar 

  36. Valoczi A, Hornyik C, Varga N et al (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32:e175

    Article  PubMed  PubMed Central  Google Scholar 

  37. Koshkin AA, Wengel J (1998) Synthesis of novel 2′,3′-linked bicyclic thymine ribonucleosides. J Org Chem 63:2778–2781

    Article  PubMed  CAS  Google Scholar 

  38. Castoldi M, Schmidt S, Benes V et al (2006) A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). RNA 12:913–920

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  39. Nelson PT, Baldwin DA, Kloosterman WP et al (2006) RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain. RNA 12:187–191

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  40. Sempere LF, Christensen M, Silahtaroglu A et al (2007) Altered microRNA expression confined to specific epithelial cell subpopulations in breast cancer. Cancer Res 67:11612–11620

    Article  PubMed  CAS  Google Scholar 

  41. Sempere LF, Preis M, Yezefski T et al (2010) Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors. Clin Cancer Res 16:4246–4255

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  42. Sempere LF, Korc M (2013) A method for conducting highly sensitive microRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer. Methods Mol Biol 980:43–59

    Article  PubMed  Google Scholar 

  43. Speel EJ, Hopman AH, Komminoth P (2006) Tyramide signal amplification for DNA and mRNA in situ hybridization. Methods Mol Biol 326:33–60

    PubMed  CAS  Google Scholar 

  44. Bobrow MN, Moen PT Jr (2001) Tyramide signal amplification (TSA) systems for the enhancement of ISH signals in cytogenetics. Curr Protoc Cytom Chapter 8, Unit

    Google Scholar 

  45. Jorgensen S, Baker A, Moller S et al (2010) Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes. Methods 52:375–381

    Article  PubMed  CAS  Google Scholar 

  46. Mardin WA, Mees ST (2009) MicroRNAs: novel diagnostic and therapeutic tools for pancreatic ductal adenocarcinoma? Ann Surg Oncol 16:3183–3189

    Article  PubMed  Google Scholar 

  47. Nielsen BS (2012) MicroRNA in situ hybridization. Methods Mol Biol 822:67–84

    Article  PubMed  CAS  Google Scholar 

  48. Singh U, Keirstead N, Wolujczyk A et al (2013) General principles and methods for routine automated microRNA in situ hybridization and double labeling with immunohistochemistry. Biotechonl Histochem 89:259–266

    Google Scholar 

  49. Pena JT, Sohn-Lee C, Rouhanifard SH et al (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  50. Liu X, Sempere LF, Guo Y et al (2011) Involvement of microRNAs in lung cancer biology and therapy. Transl Res 157:200–208

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  51. Nielsen BS, Holmstrom K (2013) Combined microRNA in situ hybridization and immunohistochemical detection of protein markers. Methods Mol Biol 986:353–365

    Article  PubMed  CAS  Google Scholar 

  52. Chaudhuri AD, Yelamanchili SV, Fox HS (2013) Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers. Front Cell Neurosci 7:160

    Article  PubMed  PubMed Central  Google Scholar 

  53. de Planell-Saguer M, Rodicio MC, Mourelatos Z (2010) Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat Protoc 5:1061–1073

    Article  PubMed  Google Scholar 

  54. Nuovo GJ (2010) In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets. Methods 52:307–315

    Article  PubMed  CAS  Google Scholar 

  55. Nuovo GJ, Elton TS, Nana-Sinkam P et al (2009) A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets. Nat Protoc 4:107–115

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  56. Nuovo GJ (2008) In situ detection of precursor and mature microRNAs in paraffin embedded, formalin fixed tissues and cell preparations. Methods 44:39–46

    Article  PubMed  CAS  Google Scholar 

  57. Hermansen SK, Dahlrot RH, Nielsen BS et al (2013) MiR-21 expression in the tumor cell compartment holds unfavorable prognostic value in gliomas. J Neurooncol 111:71–81

    Article  PubMed  CAS  Google Scholar 

  58. Yamamichi N, Shimomura R, Inada K et al (2009) Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development. Clin Cancer Res 15:4009–4016

    Article  PubMed  CAS  Google Scholar 

  59. Habbe N, Koorstra JB, Mendell JT et al (2009) MicroRNA miR-155 is a biomarker of early pancreatic neoplasia. Cancer Biol Ther 8:340–346

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  60. Preis M, Gardner TB, Gordon SR et al (2011) MicroRNA-10b Expression correlates with response to neoadjuvant therapy and survival in pancreatic ductal adenocarcinoma. Clin Cancer Res 17:5812–5821

    Google Scholar 

  61. Rask L, Balslev E, Jorgensen S et al (2011) High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer. APMIS 119:663–673

    Article  PubMed  Google Scholar 

  62. Quesne JL, Jones J, Warren J et al (2012) Biological and prognostic associations of miR-205 and let-7b in breast cancer revealed by in situ hybridization analysis of micro-RNA expression in arrays of archival tumour tissue. J Pathol 227:306–314

    Article  PubMed  CAS  Google Scholar 

  63. Kjaer-Frifeldt S, Hansen TF, Nielsen BS et al (2012) The prognostic importance of miR-21 in stage II colon cancer: a population-based study. Br J Cancer 107:1169–1174

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  64. Nielsen BS, Jorgensen S, Fog JU et al (2011) High levels of microRNA-21 in the stroma of colorectal cancers predict short disease-free survival in stage II colon cancer patients. Clin Exp Metastasis 28:27–38

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  65. Liu X, Sempere LF, Ouyang H et al (2010) MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors. J Clin Invest 120:1298–1309

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  66. Hansen TF, Andersen CL, Nielsen BS et al (2011) Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer. Oncol Lett 2:1101–1106

    PubMed  PubMed Central  Google Scholar 

  67. Gravgaard KH, Lyng MB, Laenkholm AV et al (2012) The miRNA-200 family and miRNA-9 exhibit differential expression in primary versus corresponding metastatic tissue in breast cancer. Breast Cancer Res Treat 134:207–217

    Article  PubMed  CAS  Google Scholar 

  68. Qian P, Zuo Z, Wu Z et al (2011) Pivotal role of reduced let-7g expression in breast cancer invasion and metastasis. Cancer Res 71:6463–6474

    Article  PubMed  CAS  Google Scholar 

  69. Hanna JA, Wimberly H, Kumar S et al (2012) Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization. Biotechniques 52:235–245

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  70. Hanna JA, Hahn L, Agarwal S et al (2012) In situ measurement of miR-205 in malignant melanoma tissue supports its role as a tumor suppressor microRNA. Lab Invest 92:1390–1397

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  71. Jia Z, Wang K, Zhang A et al (2013) miR-19a and miR-19b overexpression in gliomas. Pathol Oncol Res 19:847–853

    Article  PubMed  CAS  Google Scholar 

  72. Farrell JJ, Toste P, Wu N et al (2013) Endoscopically acquired pancreatic cyst fluid microRNA 21 and 221 are associated with invasive cancer. Am J Gastroenterol 108:1352–1359

    Article  PubMed  CAS  Google Scholar 

  73. Wang K, Jia Z, Zou J et al (2013) Analysis of hsa-miR-30a-5p expression in human gliomas. Pathol Oncol Res 19:405–411

    Article  PubMed  Google Scholar 

  74. Ma Y, Zhang P, Wang F et al (2012) miR-150 as a potential biomarker associated with prognosis and therapeutic outcome in colorectal cancer. Gut 61:1447–1453

    Article  PubMed  Google Scholar 

  75. Tang W, Zhu J, Su S et al (2012) MiR-27 as a prognostic marker for breast cancer progression and patient survival. PLoS One 7:e51702

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  76. Donnem T, Eklo K, Berg T et al (2011) Prognostic impact of MiR-155 in non-small cell lung cancer evaluated by in situ hybridization. J Transl Med 9:6

    Article  PubMed  PubMed Central  Google Scholar 

  77. Kadera BE, Li L, Toste PA et al (2013) MicroRNA-21 in pancreatic ductal adenocarcinoma tumor-associated fibroblasts promotes metastasis. PLoS One 8:e71978

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  78. Dillhoff M, Liu J, Frankel W et al (2008) MicroRNA-21 is overexpressed in pancreatic cancer and a potential predictor of survival. J Gastrointest Surg 12:2171–2176

    Article  PubMed  PubMed Central  Google Scholar 

  79. Renwick N, Cekan P, Masry PA et al (2013) Multicolor microRNA FISH effectively differentiates tumor types. J Clin Invest 123:2694–2702

    Article  PubMed  CAS  PubMed Central  Google Scholar 

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Acknowledgements

I would like to thank Dr. Wendy Wells for providing financial support, technical advice, and laboratory space at the Dartmouth Pathology Translation Research Laboratory, a CAP-accredited CLIA-certified facility, in which multiplex ISH/IHC assays were conducted, and Dr. Elena Bryleva for critical reading of this manuscript. This work was supported by National Institutes of Health (NIH) and National Cancer Institute (NCI) grants R21 CA141017 and R03 CA141564, and intramural Hitchcock Foundation pilot grant.

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Authors and Affiliations

  1. Program in Skeletal Disease and Tumor Microenvironment, Laboratory of microRNA Diagnostics and Therapeutics, Center for Cancer and Cell Biology, Van Andel Institute, 333 Bostwick Ave., N.E., Grand Rapids, MI, 49503, USA

    Lorenzo F. Sempere Ph.D.

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  1. Lorenzo F. Sempere Ph.D.
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Correspondence to Lorenzo F. Sempere Ph.D. .

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Editors and Affiliations

  1. Molecular Histology, Bioneer A/S, Horsholm, Denmark

    Boye Schnack Nielsen

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Sempere, L.F. (2014). Fully Automated Fluorescence-Based Four-Color Multiplex Assay for Co-detection of MicroRNA and Protein Biomarkers in Clinical Tissue Specimens. In: Nielsen, B. (eds) In Situ Hybridization Protocols. Methods in Molecular Biology, vol 1211. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1459-3_13

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  • DOI: https://doi.org/10.1007/978-1-4939-1459-3_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1458-6

  • Online ISBN: 978-1-4939-1459-3

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