Abstract
MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3’ end modified with biotin) for a target miRNA of miR-21 and capture sequence (5’ end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.
Highlights
MicroRNAs are a class of endogenous, short (19−23 nucleotides) single-stranded non-coding RNAs, which serve as critical regulators of gene expression[1]
A report probe (RP), labeled with a biotin group at the 30 terminus and a Fam group at the 50 terminus, consisting of a target miRNA recognition DNA sequence (30 end) and a capture sequence (50 end) for hybridization chain reaction (HCR) products was rationally designed, This probe was first attached to the surface of SA-coated magnetic bead (MB) through biotin-SA interaction
Upon addition of target miRNA to the reaction, it hybridized with the report probe to form a reporter probe (RP)/miRNA duplex
Summary
MicroRNAs (miRNAs) are a class of endogenous, short (19−23 nucleotides) single-stranded non-coding RNAs, which serve as critical regulators of gene expression[1] They play important roles in diverse physiological processes and diseases[2]. Conventional analytical methods, including Northern blot[6], microarrays and quantitative fluorescence reverse transcription PCR (qRT-PCR)[7], generation sequencing[8,9] are considered standard methods and widely utilized for miRNA analysis. These methods involve elaborate, time-consuming, and expensive processes that require special laboratory equipments[10]. Lateral flow nucleic acid biosensor (LFNAB) is not satisfactory for miRNA detection due to its poor sensitivity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
