Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

Weihua Guan, Liben Chen, Tushar D. Rane, Tza Huei Wang

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuousflow device. This device is capable of generating droplets at high-throughput. It also integrates inline enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

    Original languageEnglish (US)
    Article number13795
    JournalScientific reports
    Volume5
    DOIs
    StatePublished - Sep 3 2015

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    Oligonucleotides
    RNA
    Enzymes
    RNA Probes
    Equipment and Supplies
    Neisseria gonorrhoeae
    Reverse Transcription
    Polymerase Chain Reaction

    All Science Journal Classification (ASJC) codes

    • General

    Cite this

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    title = "Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification",
    abstract = "We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuousflow device. This device is capable of generating droplets at high-throughput. It also integrates inline enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40{\%} difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.",
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    Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification. / Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza Huei.

    In: Scientific reports, Vol. 5, 13795, 03.09.2015.

    Research output: Contribution to journalArticle

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    AU - Guan, Weihua

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    AU - Rane, Tushar D.

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