Real-Time Polymerase Chain Reaction MicroRNA Detection Based on Enzymatic Stem-Loop Probes Ligation

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 c...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2009-07, Vol.81 (13), p.5446-5451
Main Authors: Li, Juan, Yao, Bo, Huang, Huang, Wang, Zhao, Sun, Changhong, Fan, Yu, Chang, Qing, Li, Shaolu, Wang, Xiang, Xi, Jianzhong
Format: Article
Language:English
Subjects:
Analytical chemistry
Animals
Base Sequence
Chemistry
DNA Ligases - metabolism
DNA Probes - chemistry
DNA Probes - metabolism
Enzymes
Exact sciences and technology
Gene Amplification
Measurement
Mice
MicroRNAs - analysis
Polymerase Chain Reaction - methods
Ribonucleic acid
RNA
Rodents
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac900598d