Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification.
The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.
Silahtaroglu
Asli N
AN
Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. asli@imbg.ku.dk
Nolting
Dorrit
D
Dyrskjøt
Lars
L
Berezikov
Eugene
E
Møller
Morten
M
Tommerup
Niels
N
Kauppinen
Sakari
S
D000818:Animals; D016616:Cryoultramicrotomy; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008253:Macaca mulatta; D051379:Mice; D035683:MicroRNAs / Q000032:analysis*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D014439:Tyramine / Q000031:analogs & derivatives
C473365:FITC-tyramide; D035683:MicroRNAs; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C477371:locked nucleic acid; D016650:Fluorescein-5-isothiocyanate; D014439:Tyramine