Quantification of purified endogenous miRNAs with high sensitivity and specificity

Soochul Shin,Soomin Son,Jiyoung Goo,Sungchul Hohng,Ji-Joon Song,Soomin Son,Jiyoung Goo,Cherlhyun Jeong,Cherlhyun Jeong,Heesoo Uhm,Minseok Song,V Narry Kim,V Narry Kim,Yoonseok Jung

doi:10.1038/s41467-020-19865-9

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.

Highlights

MicroRNAs are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level
To benchmark Ago-FISH against commercialized qRT-polymerase chain reaction (PCR) techniques, we chose 5% duty cycle of probe binding as a threshold to identify as a proper target miRNA in Ago-FISH, and quantified
Compared to SiMREPS, Argonaute-based fluorescence in situ hybridization (AgoFISH) has the merits of the increased miRNA detection speed, and the conservation of the high target specificity for the whole region of miRNAs

Summary

Introduction

MicroRNAs (miRNAs) are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. We report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip. Binding of miRNAs on the spot is detected by adding fluorescently labeled streptavidin Using this technique, we can monitor a large number of target miRNAs in low cost, but the specificity and dynamic range of microarray analysis are limited because a RNA–DNA duplex of 20 base pairs is quite stable, and as a result, single base pair difference cannot be detected. We report a multicolor single-molecule imaging technique, Argonaute-based fluorescence in situ hybridization (AgoFISH), that can sequentially profile multiple purified endogenous miRNA with high sensitivity, specificity, and reliability

Methods

Results

Conclusion