Journal
ACS SENSORS
Volume 7, Issue 11, Pages 3409-3415Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.2c01526
Keywords
microRNA detection; single-molecule counting; FRET; splinted ligation; SplintR ligase
Funding
- Development of Measurement Standards and Technology for Biomaterials and Medical Convergence
- Korea Research Institute of Standards and Science
- Industrial Innovation Foundation Construction Program
- Ministry of Trade, Industry, & Energy (MOTIE, Korea)
- [KRISS-2022-GP2022-0006]
- [P0014175]
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miRNAs play a crucial role in regulating gene expression and are potential biomarkers for cancer diagnosis. A highly selective miRNA detection method using FRET technology was developed, allowing for accurate measurement of miRNA concentrations and discrimination of nucleotide mismatches.
MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Fo''rster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.
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