Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury
| Autor: | Salvatore Pernagallo, David M. Rissin, Hugh Ilyine, James W. Dear, David C. Duffy, A. D. Bastiaan Vliegenthart, Barbara López-Longarela, Juan J. Díaz-Mochón |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
B Vitamins
0301 basic medicine lcsh:Medicine Artificial Gene Amplification and Extension Biochemistry Polymerase Chain Reaction Physical Chemistry 01 natural sciences chemistry.chemical_compound Limit of Detection Chemical dynamics Amines lcsh:Science Polymerase Multidisciplinary Peptide nucleic acid biology Organic Compounds Hybridization probe Nucleic Acid Hybridization Vitamins Polymerase chain reaction Enzymes Nucleic acids Chemistry Molecular Diagnostic Techniques Biotinylation Physical Sciences Chemical and Drug Induced Liver Injury Research Article Adult Molecular Probe Techniques Biotin Research and Analysis Methods 010402 general chemistry Sensitivity and Specificity Chemical Dynamics Young Adult 03 medical and health sciences Nucleic acid thermodynamics Genetics Humans Non-coding RNA Molecular Biology Techniques Molecular Biology Acetaminophen Detection limit Chromatography Biology and life sciences Base Sequence Organic Chemistry lcsh:R Chemical Compounds Proteins Substrate (chemistry) Probe Hybridization Probe hybridization Gene regulation 0104 chemical sciences MicroRNAs Circulating MicroRNA 030104 developmental biology chemistry Case-Control Studies Enzymology biology.protein RNA lcsh:Q Gene expression Drug Overdose Biomarkers |
| Zdroj: | PLoS ONE, Vol 12, Iss 7, p e0179669 (2017) Rissin, D M, López-Longarela, B, Pernagallo, S, Ilyine, H, Vliegenthart, B, Dear, J, Díaz-Mochón, J J & Duffy, D C 2017, ' Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury ', PLoS ONE . https://doi.org/10.1371/journal.pone.0179669 Digibug. Repositorio Institucional de la Universidad de Granada instname PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0179669 |
| Popis: | 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. 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.]. |
| Databáze: | OpenAIRE |
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