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 ineffici...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2013-10, Vol.85 (19), p.9383-9389
Main Authors: Shin, Gi Won, Na, Jeongkyeong, Seo, Mihwa, Chung, Boram, Nam, Hong Gil, Lee, Seung-Jae, Jung, Gyoo Yeol
Format: Article
Language:English
Subjects:
Amplification
Analytical chemistry
Animals
Assaying
Biological
Caenorhabditis elegans - genetics
Caenorhabditis elegans - metabolism
Dynamical systems
E coli
Electrophoresis
Electrophoresis, Capillary
Escherichia coli - genetics
Escherichia coli - metabolism
Gene amplification
Genes
Kinetics
Multiplex Polymerase Chain Reaction
Multiplexing
Nematodes
Polymerase chain reaction
Profiling
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Summary: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 Escherichia coli metabolic genes and 16 Caenorhabditis elegans 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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac402314h