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