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

Abstract

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.

Scheme 1. Principle of simultaneous detection of multiple miRNAs based on the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection.

Fig. 1. (A) PAGE analysis of target miRNA-induced cyclic SDA reaction products. (B) Melting profiles of the trigger-155/AP probe-155 duplex before (black line) and after (red line) APE1 treatment. (C) Melting profiles of the trigger-21/AP probe-21 duplex before (blue line) and after (olive line) APE1 treatment. (D) Agarose gel electrophoresis analysis of target miRNA-induced RCA reaction products. (E) Normalized Cy5 fluorescence intensity in the absence (black line) and presence (red line) of miR-155. (F) Normalized Cy3 fluorescence intensity in the absence (blue line) and presence (olive line) of miR-21.

Fig. 2. (A–C) Fluorescence images of product-coupled magnetic beads in the presence of different concentrations of miR-155 and miR-21. The colocalized signals of Cy5 and Cy3 are visualized as yellow color spots (A). The red color spots (B) result from biotin-/multiple Cy5-labeled ssDNA products; the green color spots (C) result from biotin-/multiple Cy3-labeled ssDNA products. The scale bar is 2 μm. (D) Spatial distribution of the Cy5 fluorescence intensity of a single product-coupled magnetic bead. (E) Spatial distribution of the Cy3 fluorescence intensity of a single product-coupled magnetic bead.

Fig. 3. Simultaneous detection of miR-21 and miR-155 using single-molecule detection. The Cy5 fluorescence signal is shown in red (A–D), and the Cy3 fluorescence signal is shown in green (E–H). 3 nM miR-155 and 3 nM miR-21 are used in the experiments. The scale bar is 5 μm.

Fig. 4. (A) Variance of the number of Cy5 molecules with different concentrations of miR-155. The inset shows the linear relationship between the Cy5 counts and the miR-155 concentration. (B) Variance of the number of Cy3 molecules with different concentrations of miR-21. The inset shows the linear relationship between the Cy3 counts and the miR-21 concentration. Error bars show the standard deviations of three independent experiments.

Fig. 5. Measurement of Cy5 and Cy3 counts generated by miR-155, miR-21, one-base mismatched RNAs (i.e., mis-1-155 and mis-1-21), two-base mismatched RNAs (i.e., mis-2-155 and mis-2-21), three-base mismatched RNAs (i.e., mis-3-155 and mis-3-21), irrelevant miRNAs, and the reaction buffer (control). The concentration of each RNA is 30 nM. Error bars show the standard deviations of three independent experiments.

Fig. 6. Simultaneous detection of miR-155 (A, red column) and miR-21 (B, green column) in different cell lines and validation with qRT-PCR (A and B, dark gray column). The number of cells is 1000 for each cell line. Error bars show the standard deviations of three independent experiments.

Fig. 7. Simultaneous measurements of miR-155 (A, red column) and miR-21 (B, green column) in tissue samples from healthy persons and the NSCLC patients and validation with qRT-PCR (A and B, dark gray column). Error bars show the standard deviations of three independent experiments.