Abstract
MicroRNAs are short (18–23 nucleotides) noncoding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3′UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The characterization of microRNA expression in tissues by in situ detection is often crucial following a microRNA biomarker discovery phase in order to validate the candidate microRNA biomarker and allow better interpretation of its molecular functions and derived cellular interactions. The in situ hybridization data provides information about contextual distribution and cellular origin of the microRNA. By combining microRNA in situ hybridization with immunohistochemical staining of protein markers, it is possible to precisely characterize the microRNA-expressing cells and to identify the potential microRNA targets. This combined technology can also help to monitor changes in the level of potential microRNA targets in a therapeutic setting. In this chapter, we present a fluorescence-based detection method that allows the combination of microRNA in situ hybridization with immunohistochemical staining of one and, in this updated version of the paper, two protein markers detected with primary antibodies raised in the same host species.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ambros V (2001) microRNAs: tiny regulators with great potential. Cell 107:823–826
Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854
Lagos-Quintana M, Rauhut R, Lendeckel W et al (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Pillai RS, Bhattacharyya SN, Artus CG et al (2005) Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309:1573–1576
Liu J, Rivas FV, Wohlschlegel J et al (2005) A role for the P-body component GW182 in microRNA function. Nat Cell Biol 7:1261–1266
Ambros V (2011) MicroRNAs and developmental timing. Curr Opin Genet Dev 21:511–517
Cordes KR, Sheehy NT, White MP et al (2009) miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 460:705–710
Makeyev EV, Zhang J, Carrasco MA et al (2007) The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol Cell 27:435–448
Yao Q, Cao S, Li C et al (2011) Micro-RNA-21 regulates TGF-beta-induced myofibroblast differentiation by targeting PDCD4 in tumor-stroma interaction. Int J Cancer 128:1783–1792
Madhyastha R, Madhyastha H, Nakajima Y et al (2012) MicroRNA signature in diabetic wound healing: promotive role of miR-21 in fibroblast migration. Int Wound J 4:355–361
Medina PP, Slack FJ (2008) microRNAs and cancer: an overview. Cell Cycle 7:2485–2492
Winter J, Jung S, Keller S et al (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11:228–234
Betel D, Wilson M, Gabow A et al (2008) The microRNA.org resource: targets and expression. Nucleic Acids Res 36:D149–D153
Thomson DW, Bracken CP, Goodall GJ (2011) Experimental strategies for microRNA target identification. Nucleic Acids Res 39:6845–6853
Ferdin J, Kunej T, Calin GA (2010) Non-coding RNAs: identification of cancer-associated microRNAs by gene profiling. Technol Cancer Res Treat 9:123–138
Sorensen KD, Orntoft TF (2010) Discovery of prostate cancer biomarkers by microarray gene expression profiling. Expert Rev Mol Diagn 10:49–64
Jensen SG, Lamy P, Rasmussen MH et al (2011) Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs. BMC Genomics 12:435
Burnside J, Ouyang M, Anderson A et al (2008) Deep sequencing of chicken microRNAs. BMC Genomics 9:185
Joyce CE, Zhou X, Xia J et al (2011) Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome. Hum Mol Genet 20:4025–4040
Schetter AJ, Leung SY, Sohn JJ et al (2008) MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA 299:425–436
Ralfkiaer U, Hagedorn PH, Bangsgaard N et al (2011) Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL). Blood 118:5891–5900
Clop A, Marcq F, Takeda H et al (2006) A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet 38:813–818
Richardson K, Lai CQ, Parnell LD et al (2011) A genome-wide survey for SNPs altering microRNA seed sites identifies functional candidates in GWAS. BMC Genomics 12:504
Permuth-Wey J, Thompson RC, Burton NL et al (2011) A functional polymorphism in the pre-miR-146a gene is associated with risk and prognosis in adult glioma. J Neuro-Oncol 105:639–646
Lei B, Gao S, Luo LF et al (2011) A SNP in the miR-27a gene is associated with litter size in pigs. Mol Biol Rep 38:3725–3729
Nossent AY, Hansen JL, Doggen C et al (2011) SNPs in microRNA binding sites in 3′-UTRs of RAAS genes influence arterial blood pressure and risk of myocardial infarction. Am J Hypertens 24:999–1006
Zhang L, Liu Y, Song F et al (2011) Functional SNP in the microRNA-367 binding site in the 3′UTR of the calcium channel ryanodine receptor gene 3 (RYR3) affects breast cancer risk and calcification. Proc Natl Acad Sci U S A 108:13653–13658
Lodygin D, Tarasov V, Epanchintsev A et al (2008) Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 7:2591–2600
Calin GA, Ferracin M, Cimmino A et al (2005) A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 353:1793–1801
Trang P, Wiggins JF, Daige CL et al (2011) Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol Ther 19:1116–1122
Sioud M (2011) Promises and challenges in developing RNAi as a research tool and therapy. Methods Mol Biol 703:173–187
Wang Z, Rao DD, Senzer N et al (2011) RNA interference and cancer therapy. Pharm Res 12:2983–2995
Garofalo M, Croce CM (2011) microRNAs: master regulators as potential therapeutics in cancer. Annu Rev Pharmacol Toxicol 51:25–43
Gambari R, Fabbri E, Borgatti M et al (2011) Targeting microRNAs involved in human diseases: a novel approach for modification of gene expression and drug development. Biochem Pharmacol 82:1416–1429
Kasinski AL, Slack FJ (2010) Potential microRNA therapies targeting Ras, NFkappaB and p53 signaling. Curr Opin Mol Ther 12:147–157
Stenvang J, Silahtaroglu AN, Lindow M et al (2008) The utility of LNA in microRNA-based cancer diagnostics and therapeutics. Semin Cancer Biol 18:89–102
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
Soe MJ, Moller T, Dufva M et al (2011) A sensitive alternative for microRNA in situ hybridizations using probes of 2′-O-methyl RNA + LNA. J Histochem Cytochem 59:661–672
Nielsen BS (2012) MicroRNA in situ hybridization. Methods Mol Biol 822:67–84
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
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
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
Nielsen BS, Holmstrom K (2013) Combined microRNA in situ hybridization and immunohistochemical detection of protein markers. Methods Mol Biol 986:353–365
Pirici D, Mogoanta L, Kumar-Singh S et al (2009) Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype. J Histochem Cytochem 57:567–575
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
Greene SB, Herschkowitz JI, Rosen JM (2010) The ups and downs of miR-205: identifying the roles of miR-205 in mammary gland development and breast cancer. RNA Biol 7:300–304
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
Young MR, Santhanam AN, Yoshikawa N et al (2010) Have tumor suppressor PDCD4 and its counteragent oncogenic miR-21 gone rogue? Mol Interv 10:76–79
Ruan Q, Wang T, Kameswaran V et al (2011) The microRNA-21-PDCD4 axis prevents type 1 diabetes by blocking pancreatic beta cell death. Proc Natl Acad Sci U S A 108:12030–12035
Knudsen KN, Lindebjerg J, Nielsen BS et al (2017) MicroRNA-200b is downregulated in colon cancer budding cells. PLoS One 12:e0178564
Thorlacius-Ussing G, Schnack Nielsen B, Andersen V et al (2017) Expression and localization of miR-21 and miR-126 in mucosal tissue from patients with inflammatory bowel disease. Inflamm Bowel Dis 23:739–752
Sempere LF (2011) Integrating contextual miRNA and protein signatures for diagnostic and treatment decisions in cancer. Expert Rev Mol Diagn 11:813–827
Soe MJ, Okkels F, Sabourin D et al (2011) HistoFlex-a microfluidic device providing uniform flow conditions enabling highly sensitive, reproducible and quantitative in situ hybridizations. Lab Chip 11:3896–3907
Nielsen BS, Moller T, Holmstrom K (2014) Chromogen detection of microRNA in frozen clinical tissue samples using LNA probe technology. Methods Mol Biol 1211:77–84
Gould BR, Damgaard T, Nielsen BS (2017) Chromogenic in situ hybridization methods for microRNA biomarker monitoring of drug safety and efficacy. Methods Mol Biol 1641:399–412
Acknowledgments
We thank Trine Møller for excellent technical assistance and the Danish Ministry of Science, Innovation and Technology, for financial funding.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Nielsen, B.S., Holmstrøm, K. (2019). Combined MicroRNA In Situ Hybridization and Immunohistochemical Detection of Protein Markers. In: Moll, J., Carotta, S. (eds) Target Identification and Validation in Drug Discovery. Methods in Molecular Biology, vol 1953. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9145-7_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9145-7_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9144-0
Online ISBN: 978-1-4939-9145-7
eBook Packages: Springer Protocols