MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

Zhiyong Lei; Alain van Mil; Junjie Xiao; Corina H G Metz; Esther C M van Eeuwijk; Pieter A Doevendans; Joost P G Sluijter

doi:10.1016/j.btre.2018.e00255

MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

Biotechnol Rep (Amst). 2018 May 1:18:e00255. doi: 10.1016/j.btre.2018.e00255. eCollection 2018 Jun.

Authors

Zhiyong Lei¹, Alain van Mil^{1

2}, Junjie Xiao³, Corina H G Metz^{1

2}, Esther C M van Eeuwijk¹, Pieter A Doevendans^{1

4

5}, Joost P G Sluijter^{1

2}

Affiliations

¹ Department of Cardiology, Division Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands.
² UMC Utrecht Regenerative Medicine Center, University Medical Center, Utrecht 3584CT, The Netherlands.
³ Regeneration and Ageing Lab, School of Life Science, Shanghai University, Shanghai, China.
⁴ Netherlands Heart Institute (ICIN), Utrecht, The Netherlands.
⁵ Central Military Hospital, Utrecht, The Netherlands.

Abstract

To understand and assess the roles of miRNAs, visualization of the expression patterns of specific miRNAs is needed at the cellular level in a wide variety of different tissue types. Although miRNA in situ hybridization techniques have been greatly improved in recent years, they remain difficult to routinely perform due to the complexity of the procedure. In addition, as it is crucial to define which tissues or cells are expressing a particular miRNA in order to elucidate the biological function of the miRNA, incorporation of additional stainings for different cellular markers is necessary. Here, we describe a robust and flexible multicolor miRNA in situ hybridization (MMISH) technique for paraffin embedded sections. We show that the miRNA in situ protocol is sensitive and highly specific and can successfully be combined with both immunohistochemical and immunofluorescent stainings.

Keywords: Colorimetric staining; EDC, 1-Ethyl-3-[3-dimethylaminopropyl] carbodimide hydrochloride; In situ hybridization; LNA, locked nucleic acid; MI, myocardial infarction; MicroRNA; Multicolor immunofluorescence staining; NBT/BCIP, combination of nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt; Tm, melting temperature; TnI, Troponin I.