Non-radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heart.
Non-radioactive in situ hybridisation is an excellent method to visualise mRNA molecules within their topographical context. Recently we have reported a new non-radioactive in situ hybridisation procedure on tissue sections that is essentially based on the whole mount in situ hybridisation procedure. This method is superior in spatial resolution and sensitivity compared to the radioactive in situ hybridisation procedure. Generally, low levels of gene expression, such as found with the developmental onset of gene expression and in differentiating embryonic stem cells, are difficult to detect by in situ hybridisation. Here an application of the protocol is presented which is based on tyramide signal amplification, which enables the detection of very low abundant mRNAs. The significance of this method is two-fold: (1) the molecular phenotype of embryonic stem cell-derived cardiomyocytes can be examined at the cellular level with high sensitivity, and (2) the number of cells that express the gene of interest can be assessed.
Fijnvandraat
Arnoud C
AC
Experimental and Molecular Cardiology Group, Academic Medical Center, Amsterdam, The Netherlands.
De Boer
Piet A J
PA
Deprez
Ronald H Lekanne
RH
Moorman
Antoon F M
AF
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000032:analysis; D001710:Biotin / Q000031:analogs & derivatives*; D024722:Cardiac Myosins / Q000032:analysis; D006321:Heart / Q000196:embryology*; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D032386:Myoblasts, Cardiac / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000378:metabolism*; D018994:Myosin Light Chains / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D019210:Troponin I / Q000032:analysis; D014439:Tyramine / Q000031:analogs & derivatives*
D018994:Myosin Light Chains; D012333:RNA, Messenger; D019210:Troponin I; C111551:biotinyltyramide; C107184:myosin light chain 2; D001710:Biotin; D009320:Atrial Natriuretic Factor; D024722:Cardiac Myosins; D014439:Tyramine