Universidad de Granada Digibug
 

Repositorio Institucional de la Universidad de Granada >
1.-Investigación >
Departamentos, Grupos de Investigación e Institutos >
Departamento de Química Farmacéutica y Orgánica >
DQFO - Artículos >

Please use this identifier to cite or link to this item: http://hdl.handle.net/10481/47118

Title: Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury
Authors: Rissin, David M.
López-Longarela, Barbara
Pernagallo, Salvatore
Illyine, Hugh
Vliegenthart, A. D. Bastiaan
Dear, James W.
Díaz-Mochón, Juan J.
Duffy, David C.
Issue Date: 2017
Abstract: We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA) probe—containing a reactive amine instead of a nucleotide at a specific position in the sequence—for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase—containing an aldehyde group—that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG), the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122)—an established biomarker of liver toxicity—extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use.
Sponsorship: Quanterix Corporation provided support in the form of salaries and research materials for authors [D.M.R. and D.C.D.].
DestiNA Genomics Ltd. provided support in the form of salaries and research materials for authors [B.L.-L., S.P., and H.I.].
Publisher: Public Library of Science (PLOS)
Keywords: MicroRNAs
Polymerase chain reaction
Probe hybridization
Enzymes
Amines
Biomarkers
Biotin
Chemical dynamics
URI: http://hdl.handle.net/10481/47118
ISSN: 1932-6203
Rights : Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License
Citation: Rissin, D.M.; et al. Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury. Plos One, 12(7): e0179669 (2017). [http://hdl.handle.net/10481/47118]
Appears in Collections:DQFO - Artículos

Files in This Item:

File Description SizeFormat
Rissin_DrugInduced.pdf3.73 MBAdobe PDFView/Open
Recommend this item

This item is licensed under a Creative Commons License
Creative Commons

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

 

Valid XHTML 1.0! OpenAire compliant DSpace Software Copyright © 2002-2007 MIT and Hewlett-Packard - Feedback

© Universidad de Granada