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In situ hybridization with oligonucleotide probes
(With nick-translated probes)
Labeled oligonucleotides (30-50mers) can be used to detect RNAs. This method is particularly suited for the analysis of spliced vs. unspliced RNA (16, 17). The efficiency of detection is generally lower than with nick-translated probes.
Equipment and reagents
- Glass coverslips, 22x22mm
- 6-well plates
- 4% freshly made paraformaldehyde (Electron Microscopy Sciences) in PBS, pH 7.4
- Triton X-100 (Sigma)
- Parafilm
- Forceps, Dumont, GG (Electron Microscopy Sciences)
- Avidin-DCS-Texas Red or –fluorescein (Vector)
- Filterpaper
- Mounting medium (Molecular Probes)
- Microscopy coverslide
- Deionized formamide (Ambion)
- Yeast tRNA (10mg/ml) (Sigma)