title: Real-time polymerase chain reaction microRNA detection based on enzymatic stem-loop probes ligation.
abstract: MiRNAs (microRNAs) are a group of endogenous, small noncoding RNA with the length of 18-25 nucleotides, which have recently been demonstrated to play important roles in a wide range of biological processes. In this work, we developed a simple, sensitive, specific, and inexpensive assay through the combination of enzymatic probe ligation and real-time PCR amplification for the measurement of mature miRNAs. A couple of novel DNA probes with a stem-loop structure were implemented to reduce nonspecific ligation by at least 100-fold. The assay has several remarkable features including wide dynamic range, low total RNA input (0.02-0.2 ng), distinct anti-interference from precursor miRNAs (signal-to-noise ratio > 500), and single-base mismatch discrimination among miRNA sequences. In addition, a one-tube assay could be accomplished by designing a couple of universal probes, which makes it feasible to examine the expression of a whole family of miRNA (such as let-7) at one time. Finally, we validated the method for quantifying the expression of four mature miRNAs including miR-122, miR-1, miR-34a, and let-7a across 10 mouse tissues, where U6 snRNA could be simultaneously examined as an endogenous control. Thus, this method revealed a great potential for miRNA quantitation in ordinary laboratory studies and clinical diagnoses.
authors.affiliation: School of Electronic and Information Engineering, Beihang University, Beijing, China, 100191.
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Bo
authors.initials: B
authors.lastname: Yao
authors.forename: Huang
authors.initials: H
authors.lastname: Huang
authors.forename: Zhao
authors.initials: Z
authors.lastname: Wang
authors.forename: Changhong
authors.initials: C
authors.lastname: Sun
authors.forename: Yu
authors.initials: Y
authors.lastname: Fan
authors.forename: Qing
authors.initials: Q
authors.lastname: Chang
authors.forename: Shaolu
authors.initials: S
authors.lastname: Li
authors.forename: Xiang
authors.initials: X
authors.lastname: Wang
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Xi
mesh_terms: D000818:Animals; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D005784:Gene Amplification; D051379:Mice; D035683:MicroRNAs / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; C517731:Mirn1 microRNA, mouse; D011088:DNA Ligases