Multiplex quantitative analysis of microRNA expression via exponential isothermal amplification and conformation-sensitive DNA separation

Expression profiling of multiple microRNAs (miRNAs) generally provides valuable information for understanding various biological processes. Thus, it is necessary to develop a sensitive and accurate miRNA assay suitable for multiplexing. Isothermal exponential amplification reaction (EXPAR) has recei...

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Bibliographic Details
Published in:Scientific reports 2017-09, Vol.7 (1), p.11396-8, Article 11396
Main Authors: Na, Jeongkyeong, Shin, Gi Won, Son, Heehwa G., Lee, Seung-Jae V., Jung, Gyoo Yeol
Format: Article
Language:English
Subjects:
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631/1647/1513/2216
631/61/32
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Capillary electrophoresis
Conformation
Deoxyribonucleic acid
Design
DNA
DNA fingerprinting
DNA probes
Efficiency
Gene expression
Humanities and Social Sciences
Methods
MicroRNAs
miRNA
multidisciplinary
Nematodes
Polymorphism
Probes
Quantitative analysis
Science
Science (multidisciplinary)
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Summary:Expression profiling of multiple microRNAs (miRNAs) generally provides valuable information for understanding various biological processes. Thus, it is necessary to develop a sensitive and accurate miRNA assay suitable for multiplexing. Isothermal exponential amplification reaction (EXPAR) has received significant interest as an miRNA analysis method because of high amplification efficiency. However, EXPAR cannot be used for a broader range of applications owing to limitations such as complexity of probe design and lack of proper detection method for multiplex analysis. Here, we developed a sensitive and accurate multiplex miRNA profiling method using modified isothermal EXPAR combined with high-resolution capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP). To increase target miRNA specificity, a stem-loop probe was introduced instead of a linear probe in isothermal EXPAR to allow specific amplification of multiple miRNAs with minimal background signals. CE-SSCP, a conformation-dependent separation method, was used for detection. Since CE-SSCP eliminates the need for probes to have different lengths, easier designing of probes with uniform amplification efficiency was possible. Eight small RNAs comprising six miRNAs involved in Caenorhabditis elegans development and two controls were analyzed. The expression patterns obtained using our method were concordant with those reported in previous studies, thereby supporting the proposed method’s robustness and utility.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-017-11895-6