TECHNICAL NOTE Solution-phase detection of dual microRNA biomarkers in serum David Broyles & Kyle Cissell & Manoj Kumar & Sapna Deo Received: 29 June 2011 /Revised: 19 August 2011 /Accepted: 22 August 2011 /Published online: 7 September 2011 # Springer-Verlag 2011 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 complemen- tary 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 concen- trations 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. Keywords miRNA . Detection . Fluorescence . FRET . Serum Introduction Recent advances in our understanding of the vital regula- tory role that miRNA performs at both the translational and transcriptional levels have emphasized the need for faster and more reliable detection methods. This is a direct consequence of its importance in maintaining cellular homeostasis, and misregulation may be especially relevant for diseases of unknown etiology. Increasingly, researchers are demonstrating a significant correlation between abnor- mal miRNA expression levels and cancer pathogenesis in disorders such as breast cancer [1–3], colon cancer [4, 5], liver cancer [6, 7], lung cancer [8, 9], and ovarian cancer [10]. Recently, miRNA has been found to be present in bodily fluids such as serum [11, 12] and sputum [13]. Considering the short length (approximately 18–24 nucleo- tides) and low melting temperatures of miRNA, standard mRNA detection methods are often kinetically unfavorable. Current methods of miRNA detection include microarrays [14], which are excellent for high-throughput screening and profiling but are quite costly and cannot be applied in vivo. Published in the 10th Anniversary Issue. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5357-9) contains supplementary material, which is available to authorized users. D. Broyles : M. Kumar : S. Deo (*) Department of Biochemistry and Molecular Biology, University of Miami–Miller School of Medicine, 1011 NW 15th Street, Miami, FL 33136, USA e-mail: SDeo@med.miami.edu K. Cissell Department of Chemistry and Chemical Biology, Indiana University–Purdue University Indianapolis, 402 N Blackford Street, Indianapolis, IN 46202, USA Anal Bioanal Chem (2012) 402:543–550 DOI 10.1007/s00216-011-5357-9