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ŠMERKOVÁ, K. RÝPAR, T. ADAM, V. VACULOVIČOVÁ, M.
Originální název
Direct magnetic beads-based extraction of microRNA from urine with capillary electrophoretic analysis using fluorescence detection and universal label
Typ
článek v časopise ve Web of Science, Jimp
Jazyk
angličtina
Originální abstrakt
Short non-coding RNAs, specifically microRNAs (miRNAs), are of a great interest due to their presumed function in genome regulation. Moreover, miRNAs are currently perceived as potential biomarkers for numerous diseases; a variety of detection methods and sensing systems have therefore been studied. We present a magnetic-bead-based assay for specific miRNA isolation coupled with sensitive electrophoretic analysis with fluorescence detection. The magnetic separation step involves creating a duplex with targeted miR-141, which is subsequently cleaved from the magnetic bead surface with a specific endonuclease. The duplex is then determined using capillary electrophoresis with laser-induced fluorescence detection in the presence of the fluorescent dye PicoGreen for quantitating double-stranded DNA. The benefits of using microcolumn separation technique coupled with sensitive detection over traditionally used determination by fluorescence spectrometry include the fact that there is no need for a specific pre-labeled fluorescent probe. This significantly simplifies the method and reduces the costs. Cross-reactivity with mismatched oligonucleotides (3 and 5 mismatched bases) and different miRNAs (miR-124 and miR-150) was tested, demonstrating the specificity of the developed method for miRNA-141. This magnetic extraction method was demonstrated for the direct isolation and determination of miR-141 at different concentration levels from urine samples and the achieved nanomolar detection limit.
Klíčová slova
Immobilization; Sample Isolation; miR-141; Endonuclease Cleavage
Autoři
ŠMERKOVÁ, K.; RÝPAR, T.; ADAM, V.; VACULOVIČOVÁ, M.
Vydáno
31. 1. 2020
ISSN
1550-7033
Periodikum
J BIOMED NANOTECHNOL
Ročník
16
Číslo
1
Stát
Spojené státy americké
Strany od
76
Strany do
84
Strany počet
9
URL
https://doi.org/10.1166/jbn.2020.2872
BibTex
@article{BUT162416, author="Kristýna {Šmerková} and Tomáš {Rýpar} and Vojtěch {Adam} and Markéta {Vaculovičová}", title="Direct magnetic beads-based extraction of microRNA from urine with capillary electrophoretic analysis using fluorescence detection and universal label", journal="J BIOMED NANOTECHNOL", year="2020", volume="16", number="1", pages="76--84", doi="10.1166/jbn.2020.2872", issn="1550-7033", url="https://doi.org/10.1166/jbn.2020.2872" }