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Nat. Commun., Published: 27 November 2020, 11, 6033 | https://doi.org/10.1038/s41467-020-19865-9 Quantification of purified endogenous miRNAs with high sensitivity and specificity

Soochul Shin^1,7, Yoonseok Jung^2,7, Heesoo Uhm¹, Minseok Song¹, Soomin Son^2,3, Jiyoung Goo^4,5, Cherlhyun Jeong^4,5, Ji-Joon Song⁶, V. Narry Kim^2,3 & Sungchul Hohng^1,*

¹Department of Physics and Astronomy, and Institute of Applied Physics, Seoul National University, Seoul, Republic of Korea.
²Center for RNA Research, Institute for Basic Science (IBS), Seoul, Republic of Korea. ³School of Biological Sciences, Seoul National University, Seoul, Republic of Korea. ⁴Center for Theragnosis, Korea Institute of Science and Technology, Seoul, Republic of Korea.
⁵KHU-KIST Department of Converging Science and Technology, Kyunghee University, Seoul, Republic of Korea.
⁶Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.

⁷These authors contributed equally: Soochul Shin, Yoonseok Jung

^*Corresponding author

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.

논문정보

형식Research article
게재일2020년 11월 (BRIC 등록일 2020-12-07)
연구진국내 연구진
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