Authors: David Broyles Kyle Cissell Manoj Kumar Sapna Deo
Publish Date: 2011/09/07
Volume: 402, Issue: 1, Pages: 543-550
Abstract
A strategy for the simultaneous detection of multiple microRNA miRNA targets was developed utilizing fluorophore/quencherlabeled oligonucleotide probe sets Two miRNA targets miR155 and miR103 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 fluorophoreprobe and quencherprobe hybridize resulting in a fluorescence resonance energy transferbased quenching of the fluorescence signal In the presence of unlabeled target however the antisense quencherprobe can hybridize with the target resulting in increased fluorescence intensity as the quencherprobe is sequestered beyond the Förster radius of the fluorescentprobe The assay design was tested in multiple matrices of buffer cellular extract and serum and detection limits were found to be matrixdependent ranging from 034 to 889 pmol 34–593 nM for miR155 and 290–118 pmol 193–790 nM for miR103 Single double and triple nucleotide selectivity was also tested Additionally miR155 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 singlenucleotide selectivity This method can be employed for the multiplex detection of solutionphase DNA or RNA targets and more specifically for the direct detection of serum miRNA biomarkersWe would like to thank the National Science Foundation CHE0748648 and the 100 Voices of Hope project for research funding We also thank Dr Harikrishna Nakshatri and the staff of the Indiana University School of Medicine for their generous provision of clinical samples and technical assistance
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