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Journal of Molecular Histology

, Volume 42, Issue 4, pp 289–299 | Cite as

Cell-specific detection of microRNA expression during cardiomyogenesis by combined in situ hybridization and immunohistochemistry

  • Mikael Schneider
  • Ditte Caroline Andersen
  • Asli Silahtaroglu
  • Stig Lyngbæk
  • Sakari Kauppinen
  • Jakob Lerche Hansen
  • Søren Paludan Sheikh
Original Paper

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.

Keywords

MicroRNA In situ hybridization Embryoid bodies Cardiac development Heart 

Notes

Acknowledgments

We thank Mette Christensen for performing the array analysis, and Tonja Jørgensen, Sussi Mortensen and Bettina Mentz for technical help. This work was supported by The John and Birthe Meyer Foundation and The Danish Heart Foundation. Wilhelm Johannsen Centre for Functional Genome Research is established by The Danish National Research Foundation.

Supplementary material

10735_2011_9332_MOESM1_ESM.doc (45 kb)
Supplementary material 1 (DOC 45 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Mikael Schneider
    • 1
    • 2
  • Ditte Caroline Andersen
    • 1
    • 2
  • Asli Silahtaroglu
    • 3
  • Stig Lyngbæk
    • 4
  • Sakari Kauppinen
    • 3
    • 5
    • 6
  • Jakob Lerche Hansen
    • 7
    • 8
    • 9
  • Søren Paludan Sheikh
    • 1
    • 2
  1. 1.Department of Clinical Biochemistry and Pharmacology, Laboratory for Molecular and Cellular CardiologyOdense University HospitalOdenseDenmark
  2. 2.Department of Cardiovascular and Renal Research, Institute of Molecular MedicineUniversity of Southern DenmarkOdenseDenmark
  3. 3.Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular MedicineUniversity of CopenhagenCopenhagenDenmark
  4. 4.Department of CardiologyCopenhagen University HospitalGentofteDenmark
  5. 5.Santaris PharmaHørsholmDenmark
  6. 6.Copenhagen Institute of TechnologyAalborg UniversityBallerupDenmark
  7. 7.Laboratory for Molecular Cardiology, The Danish National Research Foundation Centre for Cardiac Arrhythmia, Department of Biomedical SciencesUniversity of CopenhagenCopenhagenDenmark
  8. 8.The Heart Centre, Copenhagen University HospitalRigshospitaletDenmark
  9. 9.GLP-1 and Obesity Biology, Novo NordiskMåløvDenmark

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