Quantification of purified endogenous miRNAs with high sensitivity and specificity
| Autor: | Heesoo Uhm, Ji-Joon Song, Cherlhyun Jeong, Jiyoung Goo, Soomin Son, V. Narry Kim, Yoonseok Jung, Sungchul Hohng, Soochul Shin, Minseok Song |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Science General Physics and Astronomy Endogeny Computational biology Biology 010402 general chemistry 01 natural sciences General Biochemistry Genetics and Molecular Biology Article Imaging Cell Line 03 medical and health sciences microRNA medicine Humans Differential expression Thermus Gene Multidisciplinary medicine.diagnostic_test Base Sequence General Chemistry Argonaute Thermus thermophilus biology.organism_classification 0104 chemical sciences MicroRNAs 030104 developmental biology miRNAs Imaging technique Fluorescence in situ hybridization |
| Zdroj: | Nature Communications Nature Communications, Vol 11, Iss 1, Pp 1-8 (2020) |
| ISSN: | 2041-1723 |
| Popis: | 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. MicroRNAs are potentially powerful biomarkers, though clinical use requires rapid and reliable profiling. Here the authors report amplification-free multicolour single-molecule imaging with single base mismatch sensitivity. |
| Databáze: | OpenAIRE |
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