Online ISSN : 1347-5800
Print ISSN : 0044-5991
ISSN-L : 0044-5991
An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe
Narantsog ChoijookhuuYasuaki ShibataTakumi IshizukaYan XuTakehiko KojiYoshitaka Hishikawa
Author information

2022 Volume 55 Issue 5 Pages 119-128


In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.

Content from these authors
© 2022 The Japan Society of Histochemistry and Cytochemistry

This is an open access article distributed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC-BY-NC), which permits use, distribution and reproduction of the articles in any medium provided that the original work is properly cited and is not used for commercial purposes.
Next article