Abstract

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.

Highlights

  • MicroRNAs are small non-coding RNAs that range from 18 to 25 bp in length and are found in a broad range of plants and mammals. miRNAs are known to regulate gene expression via translational repression or target degradation by hybridizing with target mRNA. miRNAs are critical regulators involved in various biological processes, such as development, differentiation, metabolism, and apoptosis

  • The results showed that our method, which is based on isothermal exponential amplification reaction (EXPAR) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP), enables highly sensitive multiplex miRNA analysis compared to qPCR-based methods

  • We demonstrate a modified isothermal EXPAR method combined with capillary electrophoresis (CE)-SSCP that can be used to quantify the expression of multiple miRNAs with high specificity using stem-loop probes

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Summary

Introduction

MicroRNAs (miRNAs) are small non-coding RNAs that range from 18 to 25 bp in length and are found in a broad range of plants and mammals. miRNAs are known to regulate gene expression via translational repression or target degradation by hybridizing with target mRNA. miRNAs are critical regulators involved in various biological processes, such as development, differentiation, metabolism, and apoptosis. Various methods have been used to investigate miRNA expression patterns, including northern blotting, quantitative RT-PCR (qRT-PCR), rolling circle amplification, loop-mediated isothermal amplification (LAMP), and isothermal exponential amplification reaction (EXPAR)[9,10,11,12,13]. None of these methods are capable of analyzing multiple miRNAs in a single assay. We developed a multiplex miRNA quantification assay that combines a modified isothermal EXPAR method and high-resolution capillary electrophoresis single strand conformation polymorphism (CE-SSCP) analysis. Eight small RNA species demonstrate the strong potential of our multiplex assay in miRNA applications

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