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Chromogenic In Situ Hybridization Methods for microRNA Biomarker Monitoring of Drug Safety and Efficacy

  • Barbara R. GouldEmail author
  • Tina Damgaard
  • Boye Schnack Nielsen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1641)

Abstract

Disease research and treatment development have turned to the impact and utility of microRNA. The dynamic and highly specific expression of these molecular regulators can be used to predict and monitor disease progression as well as therapeutic treatment efficacy and safety, thus aiding decisions in patient care. In situ hybridization (ISH) of biopsy material has become a routine clinical pathology procedure for monitoring gene structure, expression, and sample characterization. For ribonucleic acid (RNA), determining cell source and level of expression of these biomarkers gives insight into the cellular function and physiopathology. Identification and monitoring of microRNA biomarkers are made possible through locked nucleic acid (LNA)™-based detection probes. LNA™ enhances the sensitivity and specificity of target binding, most profoundly so for the short, highly similar, microRNA sequences. We present a robust 1-day ISH protocol for formalin-fixed, paraffin-embedded (FFPE) tissue sections based on microRNA-specific LNA™ detection probes which can be labeled with digoxigenin (DIG) or 6-carboxyfluorescein (FAM) and detected through enzyme-linked specific antibodies that catalyze substrates into deposited chromogen products at the target RNA site. The variety of haptens and detection reagents in combination with LNA™ chemistry offer flexibility and ease to multiple target assessment of therapeutic response.

Key words

microRNA Biomarker Locked nucleic acid Detection probe Formalin-fixed paraffin-embedded tissue 

Notes

Acknowledgments

The authors would like to thank Trine Møller at Bioneer for technical expertise as well as Marie-Louise Lunn at Exiqon for her support in the preparation of this manuscript. Exiqon is a QIAGEN company.

References

  1. 1.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297CrossRefPubMedGoogle Scholar
  2. 2.
    Wienholds E, Plasterk RH (2005) MicroRNA function in animal development. FEBS Let 579(26):5911–5922CrossRefGoogle Scholar
  3. 3.
    Li L, Chen HZ, Chen FF et al (2013) Global microRNA expression profiling reveals differential expression of target genes in 6-hybdroxydopamine-injured MN9D cells. NeuroMolecular Med 15(3):593–604CrossRefPubMedGoogle Scholar
  4. 4.
    Huang M, Lou D, Cai Q et al (2014) Characterization of paraquat-induced miRNA profiling response to hNPCs undergoing proliferation. Int J Mol Sci 15(10):18422–18436CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Su YW, Chen X, Jiang ZZ et al (2012) A panel of serum microRNAs as specific biomarkers for diagnosis of compound- and herb-induced liver injury in rats. PLoS One 7(5):e37395. doi: 10.1371/journal.pone.0037395 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wu X, Zhang Y, Xu H et al (2015) Identification of differentially expressed microRNAs involved in non-traumatic osteonecrosis through microRNA expression profiling. Gene 565(1):22–29CrossRefPubMedGoogle Scholar
  7. 7.
    Gandhi R, Healy B, Gholipour T et al (2013) Circulating microRNAs as biomarkers for disease staging in multiple sclerosis. Ann Neurol 73(6):729–740CrossRefPubMedGoogle Scholar
  8. 8.
    Anderson AL, Stanger SJ, Mihalas BP et al (2015) Assessment of microRNA expression in mouse epididymal epithelial cells and spermatozoa by next generation sequencing. Genom Data 18(6):208–211CrossRefGoogle Scholar
  9. 9.
    Ma L, Li P, Wang R et al (2015) Analysis of novel microRNA targets in drug-sensitive and—insensitive small cell lung cancer cell lines. Oncol Rep 35(3):1611–1621PubMedGoogle Scholar
  10. 10.
    Pellegrini KL, Gerlach CV, Craciun FL et al (2015) Application of small RNA sequencing to identify microRNAs in acute kidney injury and fibrosis. Toxicol Appl Pharmacol 312:42–52. doi: 10.1016/j.taap.2015.12.002 CrossRefPubMedGoogle Scholar
  11. 11.
    Koturbash I, Tolleson WH, Guo L et al (2015) microRNAs as pharmacogenomics biomarkers for drug efficacy and drug safety assessment. Biomark Med 9(11):1153–1176CrossRefPubMedGoogle Scholar
  12. 12.
    Marrone AK, Beland FA, Pogribny IP (2015) The role for microRNAs in drug toxicity and in safety assessment. Expert Opin Drug Metab Toxicol 11(4):601–611CrossRefPubMedGoogle Scholar
  13. 13.
    Thrakral S, Ghoshal K (2015) miR-122 is a unique molecule with great potential in diagnosis, prognosis of liver disease, and therapy both as a miRNA mimic and antimir. Curr Gene Ther 15(2):142–150CrossRefGoogle Scholar
  14. 14.
    Hsu J, Xu Y, Hao J et al (2012) Essential metabolic, anti-inflammatory, and anti-tumourigenic functions of miR-122 in liver. J Clin Invest 122(8):2871–2883CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Gebert LFR, Rebhan MAE, Crivelli SEM et al (2014) Miravirsen (SPC3649) can inhibit the biogenesis of miR-122. Nucleic Acids Res 42(1):609–621CrossRefPubMedGoogle Scholar
  16. 16.
    Zhao Y-N, Li W-F, Zhang Z et al (2013) Resveratrol improves learning and memory in normally aged mice through microRNA-CREB pathway. Biochem Biophys Res Commun 435(4):597–602CrossRefPubMedGoogle Scholar
  17. 17.
    Kontaraki JE, Marketou ME, Zacharis EA et al (2014) MicroRNA-9 and microRNA-126 expression levels in patients with essential hypertension: potential markers of target-organ damage. J Am Soc Hypertens 8(6):368–375CrossRefPubMedGoogle Scholar
  18. 18.
    Yan T, Cui K, Huang X et al (2014) Assessment of therapeutic efficacy of miR-126 with contrast-enhanced ultrasound in preeclampsia rats. Placenta 35(1):23–29CrossRefPubMedGoogle Scholar
  19. 19.
    Cheng Y, Liu X, Yang J et al (2009) MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neoinimal lesion formation. Circ Res 105(2):158–166CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Boettger T, Beetz N, Kostin S et al (2009) Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster. J Clin Invest 119(9):2634–2647CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Chivukula RR, Shi G, Acharya A et al (2014) An essential mesenchymal function for miR-143/145 in intestinal epithelial regeneration. Cell 157(5):1104–1116CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Wienholds E, Kloosterman WP, Miska E et al (2005) MicroRNA expression in zebrafish embryonic development. Science 309(5732):310–311CrossRefPubMedGoogle Scholar
  23. 23.
    Vester B, Wengel J (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43(42):13233–13241CrossRefPubMedGoogle Scholar
  24. 24.
    Jorgensen S, Baker A, Møller S et al (2010) Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes. Methods 52(4):375–381CrossRefPubMedGoogle Scholar
  25. 25.
    Nielsen BS (2012) MicroRNA in situ hybridization. Methods Mol Biol 822:67–84CrossRefPubMedGoogle Scholar
  26. 26.
    Nielsen BS, Møller T, Holmstrøm K (2014) Chromogen detection of microRNA in frozen clinical tissue samples using LNA™ probe technology. Methods Mol Biol 1211:77–84CrossRefPubMedGoogle Scholar
  27. 27.
    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. Biotech Histochem 89(4):259–266CrossRefPubMedGoogle Scholar
  28. 28.
    Toledano H, D’Alterio C, Loza-Coll M et al (2012) Dual fluorescence detection of protein and RNA in Drosophila tissues. Nat Protoc 7(10):1808–1817CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Keeney JG, Davis JM, Siegenthaler J et al (2015) DUF1220 protein domains drive proliferation in human neural stem cells and are associated with increased cortical volume in anthropoid primates. Brain Struct Funct 220(5):3053–3060CrossRefPubMedGoogle Scholar
  30. 30.
    Nielsen BS, Holmstrøm K (2013) Combined microRNA in situ hybridization and immunohistochemical detection of protein markers. Methods Mol Biol 986:353–365CrossRefPubMedGoogle Scholar
  31. 31.
    Sempere LF, Peris M, Yezefski T et al (2010) Fluorescence-based co-detection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors. Clin Cancer Res 16(16):4246–4255CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Darnell DK, Stanislaw S, Kaur S et al (2010) Whole mount in situ hybridization detection of mRNAs using short LNA containing DNA oligonucleotide probes. RNA 16:632–637CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Exiqon (2016) miRCURY LNA™ microRNA ISH Optimization Kit (FFPE) manual. V.3 http://www.exiqon.com/ls/Documents/Scientific/miRCURY-LNA-microRNA-ISH-Optimization-Kit-manual.pdf
  34. 34.
    Nielsen BS, Jørgensen 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 28:27–38CrossRefPubMedGoogle Scholar
  35. 35.
    Knudsen KN, Nielsen BS, Lindebjerg J et al (2015) microRNA-17 is the most up-regulated member of the miR-17-92 cluster during early colon cancer evolution. PLoS One 10(10):e0140503. doi:10.1371/journal. pone.0140503CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Mestdagh P, Hartmann N, Baeriswyl L et al (2014) Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study. Nat Methods 11(8):809–815CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Barbara R. Gould
    • 1
    Email author
  • Tina Damgaard
    • 2
  • Boye Schnack Nielsen
    • 3
  1. 1.Exiqon Inc.WoburnUSA
  2. 2.Exiqon A/SVedbækDenmark
  3. 3.Bioneer A/SHørsholmDenmark

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