Quantification of purified endogenous miRNAs with high sensitivity and specificity

Soochul Shin, Yoonseok Jung, Heesoo Uhm, Minseok Song, Soomin Son, Jiyoung Goo, Cherlhyun Jeong, Ji-Joon Song, V. Narry Kim and Sungchul Hohng ()
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Soochul Shin: Seoul National University
Yoonseok Jung: Center for RNA Research, Institute for Basic Science (IBS)
Heesoo Uhm: Seoul National University
Minseok Song: Seoul National University
Soomin Son: Center for RNA Research, Institute for Basic Science (IBS)
Jiyoung Goo: Center for Theragnosis, Korea Institute of Science and Technology
Cherlhyun Jeong: Center for Theragnosis, Korea Institute of Science and Technology
Ji-Joon Song: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST)
V. Narry Kim: Center for RNA Research, Institute for Basic Science (IBS)
Sungchul Hohng: Seoul National University

Nature Communications, 2020, vol. 11, issue 1, 1-8

Abstract: Abstract MicroRNAs (miRNAs) are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3′-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.

Date: 2020
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DOI: 10.1038/s41467-020-19865-9

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