Solution-phase detection of dual microRNA biomarkers in serum

David Broyles, Kyle Cissell, Manoj Kumar, Sapna Deo

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)543-550
Number of pages8
JournalAnalytical and bioanalytical chemistry
Volume402
Issue number1
DOIs
StatePublished - Jan 1 2012

Keywords

  • Detection
  • Fluorescence
  • FRET
  • miRNA
  • Serum

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

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