Cell-specific detection of microRNA expression during cardiomyogenesis by combined in situ hybridization and immunohistochemistry |
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Authors: | Mikael Schneider Ditte Caroline Andersen Asli Silahtaroglu Stig Lyngbæk Sakari Kauppinen Jakob Lerche Hansen Søren Paludan Sheikh |
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Institution: | 1.Department of Clinical Biochemistry and Pharmacology, Laboratory for Molecular and Cellular Cardiology,Odense University Hospital,Odense,Denmark;2.Department of Cardiovascular and Renal Research, Institute of Molecular Medicine,University of Southern Denmark,Odense,Denmark;3.Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular Medicine,University of Copenhagen,Copenhagen,Denmark;4.Department of Cardiology,Copenhagen University Hospital,Gentofte,Denmark;5.Santaris Pharma,H?rsholm,Denmark;6.Copenhagen Institute of Technology,Aalborg University,Ballerup,Denmark;7.Laboratory for Molecular Cardiology, The Danish National Research Foundation Centre for Cardiac Arrhythmia, Department of Biomedical Sciences,University of Copenhagen,Copenhagen,Denmark;8.The Heart Centre, Copenhagen University Hospital,Rigshospitalet,Denmark;9.GLP-1 and Obesity Biology, Novo Nordisk,M?l?v,Denmark |
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Abstract: | MicroRNAs (miRNAs) regulate gene expression by mediating translational repression or mRNA degradation of their targets, and
several miRNAs control developmental decisions through embryogenesis. In the developing heart, miRNA targets comprise key
players mediating cardiac lineage determination. However, although several miRNAs have been identified as differentially regulated
during cardiac development and disease, their distinct cell-specific localization remains largely undetermined, likely owing
to a lack of adequate methods. We therefore report the development of a markedly improved approach combining fluorescence-based
miRNA-in situ hybridization (miRNA-ISH) with immunohistochemistry (IHC). We have applied this protocol to differentiating
embryoid bodies (EBs) as well as embryonic and adult mouse hearts, to detect miRNAs that were upregulated during EB cardiomyogenesis,
as determined by array-based miRNA expression profiling. In this manner, we found specific co-localization of miR-1 to myosin
positive cells (cardiomyocytes) of EBs, developing and mature hearts. In contrast, miR-125b and -199a did not localize to
cardiomyocytes, as previously suggested for miR-199a, but were rather expressed in connective tissue cells of the heart. More
specifically, by co-staining with α-smooth muscle actin (α-SMA) and collagen-I, we found that miR-125b and -199a localize
to perivascular α-SMA− stromal cells. Our approach thus proved valid for determining cell-specific localization of miRNAs, and the findings we present
highlight the importance of determining exact cell-specific localization of miRNAs by sequential miRNA-ISH and IHC in studies
aiming at understanding the role of miRNAs and their targets. This approach will hopefully aid in identifying relevant miRNA
targets of both the heart and other organs. |
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