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 language | English (US) |
|---|---|
| Article number | 13795 |
| Journal | Scientific reports |
| Volume | 5 |
| DOIs | |
| State | Published - Sep 3 2015 |
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All Science Journal Classification (ASJC) codes
- General
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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 journal › Article
TY - JOUR
T1 - Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification
AU - Guan, Weihua
AU - Chen, Liben
AU - Rane, Tushar D.
AU - Wang, Tza Huei
PY - 2015/9/3
Y1 - 2015/9/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84940870090&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84940870090&partnerID=8YFLogxK
U2 - 10.1038/srep13795
DO - 10.1038/srep13795
M3 - Article
C2 - 26333806
AN - SCOPUS:84940870090
VL - 5
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
M1 - 13795
ER -