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 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.113sciescopu

Simultaneous Determination of Multiple microRNA Levels Utilizing Biotinylated Dideoxynucleotides and Mass Spectrometry.

MicroRNAs (miRNAs) are important regulators of gene translation and have been suggested as potent biomarkers in various disease states. In this study, we established an efficient method for simultaneous determination of multiple miRNA levels, employing the previously developed SPC-SBE (solid phase capture-single base extension) approach and MALDI-TOF mass spectrometry (MS). In this approach, we first perform reverse transcription of miRNAs extracted using stem-loop primers. Then the cDNA is co-amplified with competitors, synthetic oligonucleotides whose sequences precisely match cDNA except for one base, and the amplicons serve as templates for a multiplexed SBE reaction. Extension products are isolated using SPC and quantitatively analyzed with MALDI-TOF MS to determine multiple miRNA levels. Here we demonstrated concurrent analysis of four miRNA levels utilizing the approach. Furthermore, we showed the presented method significantly facilitated MS analysis of peak area ratio owing to SPC. The SPC process allowed effective removal of irrelevant reaction components prior to MS and promoted MS sample purification. Data obtained in this study was verified with RT-qPCR and agreement was shown on one order of magnitude scale, suggesting the SPC-SBE and MS approach has strong potential as a viable tool for high throughput miRNA analysis

Sequences of stem-loop RT primers (upper) and competitors (lower).

<p>Sequences of stem-loop RT primers (upper) and competitors (lower).</p

SBE (Single Base Extension) primers.

<p>SBE (Single Base Extension) primers.</p

Peak area ratios measured from MS, calculated concentrations of miRNAs in cPCR reaction, and relative levels of miRNA measured in RT-qPCR.

<p>Peak area ratios measured from MS, calculated concentrations of miRNAs in cPCR reaction, and relative levels of miRNA measured in RT-qPCR.</p

Mass spectrum for multiplexed analysis of four miRNA levels.

<p>Levels of cellular and spiked miRNA are determined using the peak area ratio and the initial concentration of the corresponding competitors.</p

The SPC-SBE and MS approach for multiplexed miRNA quantification.

<p>Reverse transcription of miRNA using stem-loop primers is followed by co-amplification of cNDA and competitors with single base alterations. A library of SBE primers with distinct masses are extended by biotin-ddNTPs in a multiplexed SBE reactions. Two extension products are generated for each SBE primer, one for miRNA and one for its competitor. Then the extension products are purified in an SPC process and analyzed by MALDI-TOF MS. Area ratio of extension product peaks is used to determine the levels of miRNAs.</p

Precise Expression Profiling by Stuffer-Free Multiplex Ligation-Dependent Probe Amplification

In systems biological studies, precise expression profiling of functionally important gene sets is crucial. Real-time polymerase chain reaction is generally used for this purpose. Despite its widespread acceptance, however, this method is not suitable for multiplex analysis, resulting in an inefficient assay process. One alternative technology in the spotlight is multiplex ligation-dependent probe amplification (MLPA). But MLPA depends on length-based discrimination of amplified products, which complicates probe design and compromises analysis results. Here, we devised a variation of MLPA that utilizes conformation-sensitive capillary electrophoresis, and demonstrated the simplicity of the probe-design process and improved precision of the assay in analyses of 33 <i>Escherichia coli</i> metabolic genes and 16 <i>Caenorhabditis elegans</i> longevity-related genes. The results showed that relative expression could be quantitatively measured over a relevant dynamic range by using similar-sized probes. Importantly, the improved precision compared to conventional MLPA promises a wider application of this method for various biological systems