Multicolor fluorescence encoding of different microRNAs in lung cancer tissues at the single-molecule level

The simultaneous detection of multiple microRNAs (miRNAs) will facilitate early clinical diagnosis. Herein, we demonstrate the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection for simultaneous measurement of different miRNAs in lung cancer tissues. This assay involves two linear templates and two circular templates without the requirement of any fluorescent-labeled probes. The binding of target miRNAs to their corresponding linear templates initiates the cyclic strand displacement amplification, generating many triggers which can specifically hybridize with the corresponding biotin-labeled AP probes to initiate the apurinic/apyrimidic endonuclease 1-assisted cyclic cleavage reaction for the production of more biotin-labeled primers for each miRNA. The resultant two primers can react with their corresponding circular templates to initiate rolling circle amplification which enables the incorporation of Cy5-dCTP/Cy3-dGTP nucleotides, resulting in the simultaneous production of abundant biotin-/multiple Cy5/Cy3-labeled DNA products. After magnetic separation and exonuclease cleavage, the amplified products release abundant Cy5 and Cy3 fluorescent molecules which can be simply monitored by single-molecule detection, with Cy3 indicating miR-21 and Cy5 indicating miR-155. This assay involves three consecutive amplification reactions, enabling the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 fluorophore-encoded DNA products which can release abundant fluorescent molecules for the generation of amplified signals. This assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay. This method can simultaneously quantify miR-155 and miR-21 in living cells and in lung cancer tissues, and it can distinguish the expression of miRNAs between non-small cell lung cancer patients and healthy persons. The accuracy and reliability of the proposed method are further validated by quantitative reverse transcription polymerase chain reaction.