Fluorescence resonance energy transfer-based hybridization chain reaction for in situ visualization of tumor-related mRNA

Abstract

The ability to visualize tumor-related mRNA in situ in single cells would distinguish whether they are cancer cells or normal cells, which holds great promise for cancer diagnosis at an early stage. Fluorescence resonance energy transfer (FRET) and hybridization chain reactions (HCRs) were combined with amplified sense tumor-related mRNA (TK1 mRNA) in situ with high sensitivity in single cells and tissue sections. Using this strategy, each copy of the target mRNA can propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked duplex that contains repeated FRET units, amplifying the fluorescent signal. The detection limit of 18 pM is about three orders of magnitude lower than that of a non-HCR method (such as the binary-probe-system). Meanwhile, due to the FRET strategy, complicated washing steps are not necessary and experimental time is sharply reduced. As far as we know, this is the first report of a fluorescence in situ hybridization (FISH) strategy that can simultaneously fulfil signal amplification and is wash-free. We believe that this FRET-based HCR strategy has great potential as a powerful tool in basic research and clinical diagnosis.