Direct Quantification of Single-Molecules of MicroRNA by Total Internal Reflection Fluorescence Microscopy

Abstract

MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT-PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis.

A single quantum dot-based biosensor for DNA point mutation assay

“…In order to minimize the photobleaching, an oxygen-scavenging buffer (1 mg/mL glucose oxidase, 0.4 % (w/v) D-glucose, and 0.04 mg/mL catalase) was added into the imaging buffer. The glass slides were pretreated according to the method proposed by Chan et al 31 The H460 cells were cultured in Dulbecco's modified Eagle's media (DMEM) supplemented with 10 % fetal bovine serum, 100 μg/mL penicillin and 100 μg/mL streptomycin. The cells were incubated at 37 °C in humidified air with 5 % CO The QDs were excited by a Jive 488nm DPSS laser (Cobolt) via TIRF.…”

Simultaneous Measurement of SUMOylation using SNAP/CLIP‐Tag‐Mediated Translation at the Single‐Molecule Level

“…[43] Received: August 18, 2012 Published online: November 21, 2012 . A sapphire 488 nm laser (50 mW, Coherent, USA) and a cube 640 nm laser (100 mW, Coherent, USA) were used for the excitation of ATTO 488 and Alexa Fluor 647.…”

“…The number of fluorescent molecules were determined according to a reported method. [43] Received: August 18, 2012 Published online: November 21, 2012 . Keywords: fluorescence · protein modifications · single-molecule studies · SNAP tag · SUMOylation…”

Functionalized h‐BN Nanosheets as a Theranostic Platform for SERS Real‐Time Monitoring of MicroRNA and Photodynamic Therapy

“…The ratiometric determination was designed with built‐in corrections for avoiding environmental effects. In this way, the developed nanoplatform with ultrasensitivity, high selectivity, and accuracy was employed for miR‐21 monitoring and imaging with limit of detection (LOD) down to 0.7 f m in live cells, which was 100 times lower than that of the previous methods . On the other hand, CuPc intercalated into HG DNA improved its solubility to act as an effective PS for PDT.…”