MicroRNA detection in bone marrow cells by LNA-FISH.
The protocol reported in this chapter describes a method for the detection and spatial localisation of microRNAs (miRNAs) in cryopreserved primary leukaemic suspension cells using digoxigenin (DIG)-labelled, Locked Nucleic Acid (LNA)-modified probes, and fluorescence in situ hybridisation (FISH). The LNA probe hybridisation yields highly accurate signals able to discriminate between single nucleotide differences and hence between closely related miRNA family members. DIG-labelled LNA probes for mature miRNAs are detected using an anti-DIG fluorescein isothiocyanate (FITC) conjugated antibody and the fluorescent signals visualised with a confocal microscope, which permits the spatial localisation of the miRNAs. Using LNA-FISH, we visualised the spatial localisation of two mature miRNAs, miR-127 and miR-154, in primary acute myeloid leukaemia (AML) suspension cells, and thus, we confirmed their expression in a specific leukaemic subtype as measured by real-time PCR.
Debernardi
Silvana
S
Medical Oncology Centre, Barts & The London School of Medicine and Dentistry, Institute of Cancer, Queen Mary University of London, London, UK.
Dixon-McIver
Amanda
A
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation / Q000379:methods; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D014407:Tumor Cells, Cultured
D035683:MicroRNAs; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C477371:locked nucleic acid