Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections.
Here we describe a method for sensitive and specific histological detection of microRNAs (miRNAs) by in situ hybridization. The protocol focuses on the use of locked nucleic acids (LNAs), which are bi-cyclic RNA analogs that allow a significant increase in the hybridization temperature and thereby an enhanced stringency for short probes as required for miRNA detection. The protocol is optimized for cryosections in order to study the spatial and temporal expression of miRNAs with high sensitivity and resolution. We detail how to construct probes, set up and conduct an LNA in situ hybridization experiment. In addition, we discuss alternative colorimetric strategies that can be used to effectively detect and visualize miRNAs including double staining with other markers. Setting up and conducting the in situ experiment is estimated to take approximately 1 week, assuming that all the component parts are readily available.
Obernosterer
Gregor
G
Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr Bohr-Gasse 3, 1030 Vienna, Austria.
Martinez
Javier
J
Alenius
Mattias
M
D000818:Animals; D005786:Gene Expression Regulation; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015336:Molecular Probe Techniques; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity
D035683:MicroRNAs; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid