Determination of absolute expression profiles using multiplexed miRNA analysis

Yunke Song, Duncan Kilburn, Jee Hoon Song, Yulan Cheng, Christopher T. Saeui, Douglas G. Cheung, Carlo M. Croce, Kevin J Yarema, Stephen Meltzer, Kelvin J. Liu, Tza Huei Wang

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Abstract

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR's ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes.

Original languageEnglish (US)
Article numbere0180988
JournalPLoS One
Volume12
Issue number7
DOIs
StatePublished - Jul 1 2017

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ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Song, Y., Kilburn, D., Song, J. H., Cheng, Y., Saeui, C. T., Cheung, D. G., Croce, C. M., Yarema, K. J., Meltzer, S., Liu, K. J., & Wang, T. H. (2017). Determination of absolute expression profiles using multiplexed miRNA analysis. PLoS One, 12(7), [e0180988]. https://doi.org/10.1371/journal.pone.0180988