title: Magnetic bead-based hybridization assay for electrochemical detection of microRNA.
abstract: Aberrant expression of microRNAs (miRNAs), short non-coding RNA molecules regulating gene expression, is often found in tumor cells, making the miRNAs suitable candidates as cancer biomarkers. Electrochemistry is an interesting alternative to current standard methods of miRNA detection by offering cheaper instrumentation and faster assays times. In this paper, we labeled miRNA in a quick, simple, two-step procedure with electroactive complex of osmium(VI) and 2,2'-bipyridine, Os(VI)bipy, which specifically binds to the ribose at the 3'-end of the miRNA, and hybridized such labeled miRNA with biotinylated capture probe attached to the streptavidin magnetic beads. Labeled miRNA was then detected at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of the peak from the Os(VI)bipy label. We obtained good selectivity of the assay using elevated hybridization temperatures for better discrimination of perfect duplex from single and double mismatches. After optimization of the protocol, we demonstrated feasibility of our assay by detecting target miRNA in real total RNA samples isolated from human cancer cells.
authors: Regional Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic; Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 612 65 Brno, Czech Republic. Electronic address: martin.bartosik@mou.cz.
authors: Martin
authors: M
authors: Bartosik
authors: Regional Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic.
authors: Roman
authors: R
authors: Hrstka
authors: Regional Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic; Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 612 65 Brno, Czech Republic.
authors: Emil
authors: E
authors: Palecek
authors: Regional Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic.
authors: Borivoj
authors: B
authors: Vojtesek
mesh_terms: D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods; D019921:Biotinylation; D055664:Electrochemical Techniques / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D060328:Magnetic Phenomena; D008628:Mercury / Q000737:chemistry; D035683:MicroRNAs / Q000032:analysis*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009942:Organometallic Compounds / Q000737:chemistry
chemical_list: D035683:MicroRNAs; D009942:Organometallic Compounds; D015082:2,2'-Dipyridyl; C076491:osmium tetroxide-2,2'-bipyridine; D008628:Mercury
keywords: Electrochemistry; Mercury electrodes; MicroRNA; Mismatch discrimination