Detection of specific miRNAs in a sample is often done in the lab using qPCR, microarray or deep sequencing methods. For point-of-care purposes, it is desirable to develop faster and cheaper miRNA detection methods. In a recent edition of Chemical Communications, authors Guo et al. describe a method to detect multiple miRNAs in a sample using fluorescent beads, specific probes and a fluorescent intercalating dye.
Briefly, “barcoded” beads were prepared with different ratios of blue and green quantum dot dyes embedded inside, and coated with silica to improve quantum dot stability. Each bead barcode population was conjugated to a specific miRNA capture probe, which was designed to form a stem-loop structure when unbound. In the presence of the target miRNA the probe and target hybridize, freeing the stem loop portion of the probe up to hybridize with a series of overlapping extension oligos. Therefore, when the capture beads are incubated in the presence of target miRNA and extension oligos, a long dsDNA chain will form, attached to the bead. The beads are then incubated with the fluorescent DNA intercalating dye, GelRed®, to label those beads that formed the dsDNA extension (and therefore the beads that bound the target miRNA).
For each barcoded sample, the authors calculated the limit of detection to be 70 fM of miRNA, and the dynamic range to be 0.15 pM to 3 nM. They also tested the assay specificity and found that a single-base mismatch in the miRNA was enough to reduce the signal 4-fold, demonstrating that the assay is quite sequence-specific. Finally, they combined four barcoded beads and incubated them in cell lysates to try to detect endogenous miRNAs miR-21, miR-10b, miR-155 and miR-373. They determined that 1000 cells was sufficient for good detection of endogenous miRNA using this Qbead-GelRed® method.
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Guo, Qingsheng and Bian, Feika and Liu, Yuqian and Qu, Xiaojun and Hu, Xianyun and Sun, Qingjiang. Hybridization chain reactions on silica coated Qbeads for the colorimetric detection of multiplex microRNAs. Chem. Commun. 2017. 53, 36, pp 4954-4957; doi: 10.1039/C7CC00462A
Learn more about the safe, bright DNA-binding dye, GelRed®. Click here.