title: Solution-phase detection of dual microRNA biomarkers in serum.
abstract: A strategy for the simultaneous detection of multiple microRNA (miRNA) targets was developed utilizing fluorophore/quencher-labeled oligonucleotide probe sets. Two miRNA targets (miR-155 and miR-103), whose misregulation has afforded them status as putative biomarkers in certain types of cancer, were detected using our assay design. In the absence of target, the complementary fluorophore-probe and quencher-probe hybridize, resulting in a fluorescence resonance energy transfer-based quenching of the fluorescence signal. In the presence of unlabeled target, however, the antisense quencher-probe can hybridize with the target, resulting in increased fluorescence intensity as the quencher-probe is sequestered beyond the Förster radius of the fluorescent-probe. The assay design was tested in multiple matrices of buffer, cellular extract, and serum; and detection limits were found to be matrix-dependent, ranging from 0.34 to 8.89 pmol (3.4-59.3 nM) for miR-155 and 2.90-11.8 pmol (19.3-79.0 nM) for miR-103. Single, double, and triple nucleotide selectivity was also tested. Additionally, miR-155 concentrations were assessed in serum samples obtained directly from breast cancer patients without the need for RNA extraction. This assay is quantitative, possesses a low detection limit, can be applied in multiple complex matrices, and can obtain single-nucleotide selectivity. This method can be employed for the multiplex detection of solution-phase DNA or RNA targets and, more specifically, for the direct detection of serum miRNA biomarkers.
authors: Department of Biochemistry and Molecular Biology, University of Miami-Miller School of Medicine, Miami, FL 33136, USA.
authors: David
authors: D
authors: Broyles
authors: Kyle
authors: K
authors: Cissell
authors: Manoj
authors: M
authors: Kumar
authors: Sapna
authors: S
authors: Deo
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000097:blood; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000097:blood*; D015345:Oligonucleotide Probes / Q000097:blood*
chemical_list: D015415:Biomarkers; D035683:MicroRNAs; D015345:Oligonucleotide Probes