Figure 4.

LNA-based in situ detection of miRNAs: keys to successful hybridization. The placenta contains high-copy miR-130¹⁹. In situ hybridization for miR-130 shows an intense signal (brown, NBT/BCIP) with a probe concentration of 0.4 pmol μl⁻¹ if the post-hybridization wash is at (a) 4 °C or (b) 50 °C. The inset shows no signal with the scrambled probe under the same conditions of either panel a or b. In comparison, the copy number of miR-130 is 93% less in the brain compared with the placenta¹⁹. Panel c shows a signal in the brain at a probe concentration of 0.05 pmol μl⁻¹ if the post-hybridization wash is at 4 °C but no signal if the post-hybridization wash is at 50 °C, (d) even if the probe concentration is increased to 0.8 pmol ml⁻¹. The inset in panel d shows that, under similar conditions, a strong signal is seen in mouse embryo if the stem cell marker miR-302 probe is used at a concentration of 0.4 pmol μl⁻¹. By combining RT in situ PCR for the precursor with LNA in situ hybridization, one can determine that benign tissues (such as the leiomyoma in panel e) can show miR-221 precursors (blue signal) and not the mature form (inset—LNA probe where no signal was evident); note the nuclear localization of the miRNA precursor. (f) When the mature form of miR-221 is evident, as is typical in this invasive leiomyosarcoma, the miR-221 precursor (RTISPCR) and mature miR-221 signal from the LNA probe both tend to localize to the cytoplasm (each panel is at ×400 original magnification).