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

Scientific Reports
Weihua GuanTza-Huei Wang

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 continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line 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.

References

Oct 10, 1997·Nucleic Acids Research·W HenkeS A Loening
Aug 4, 1999·Proceedings of the National Academy of Sciences of the United States of America·B Vogelstein, K W Kinzler
Jan 17, 2004·Clinical Chemistry·Anders StåhlbergMikael Kubista
Nov 14, 2006·Proceedings of the National Academy of Sciences of the United States of America·Luigi WarrenStephen R Quake
Apr 5, 2007·Nature Protocols·Tania NolanStephen A Bustin
Feb 19, 2008·Nature Biotechnology·Gary K GeissKrassen Dimitrov
Oct 17, 2008·Nature·Edwin H Cook, Stephen W Scherer
Jul 5, 2011·Nature Methods·Kevin A HeyriesCarl L Hansen
Oct 1, 2011·Lab on a Chip·Andrew C HatchAbraham P Lee
Oct 11, 2011·Journal of Virological Methods·Richard Allen WhiteKenneth Curr
Nov 1, 2011·Analytical Chemistry·Benjamin J HindsonBill W Colston
Feb 23, 2012·Nature Chemistry·David Yu ZhangPeng Yin
Apr 19, 2012·Nature Reviews. Genetics·Colin C PritchardMuneesh Tewari
Jul 31, 2012·Lab on a Chip·Tushar D RaneTza-Huei Wang
Sep 19, 2012·Nature Reviews. Genetics·Christoph Bock
Apr 3, 2013·Lab on a Chip·Rerngchai ArayanarakoolJan C T Eijkel
May 17, 2013·Scientific Reports·Tobias KlampMarkus Sauer
Sep 28, 2013·PloS One·Rebecca SandersJim F Huggett
Nov 28, 2013·Analytical and Bioanalytical Chemistry·Nejc RačkiMaja Ravnikar
Feb 5, 2014·Journal of the American Chemical Society·Federica DegliangeliRoberto Fiammengo
Feb 21, 2014·Science Translational Medicine·Chetan BettegowdaLuis A Diaz
Jun 14, 2014·Frontiers in Oncology·Kenji TakahashiTushar Patel
Nov 29, 2014·Lab on a Chip·Tushar D RaneTza-Huei Wang

❮ Previous
Next ❯

Citations

Jan 29, 2016·Lab on a Chip·Sarah M FriedrichTza-Huei Wang
May 12, 2018·Small·Jayson V PagaduanDavid H Gracias
May 26, 2018·Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology·Aniruddha M KaushikTza-Huei Wang
Nov 3, 2020·Clinical and Translational Medicine·Steven D HicksFrank Middleton
Jun 12, 2018·Nano Letters·Chunchen LiuLei Zheng
Jul 20, 2019·Analytical Chemistry·Reza NouriWeihua Guan
May 26, 2017·Chemical Reviews·Luoran ShangYuanjin Zhao

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
chip
chips
reverse transcription PCR
electrophoresis
Assay

Software Mentioned

ELOHA
LabVIEW
MATLAB

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.