Research Article: Determination of absolute expression profiles using multiplexed miRNA analysis

Date Published: July 13, 2017

Publisher: Public Library of Science

Author(s): Yunke Song, Duncan Kilburn, Jee Hoon Song, Yulan Cheng, Christopher T. Saeui, Douglas G. Cheung, Carlo M. Croce, Kevin J. Yarema, Stephen J. Meltzer, Kelvin J. Liu, Tza-Huei Wang, Sebastien Pfeffer.


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.

Partial Text

MicroRNA (miRNA) are short (18–24 nt) non-coding RNA molecules that regulate gene expression. They exert control over a wide variety of cellular processes, ranging from differentiation to growth and senescence [1–5]. Although over 2000 human miRNA are predicted to exist, individual studies often focus on smaller subsets of 5–50 miRNA that are the presumed critical players in a specific disease or cellular process [3,6–8]. These miRNA are typically identified by differential expression analysis using highly multiplexed array methods [9–12] or RNA sequencing [13–15]. Validation of identified miRNA is then performed using a higher sensitivity detection platform such as RT-qPCR [16–18]. However, screening large numbers of samples across even moderate numbers of miRNA using qRT-PCR quickly becomes cost- and time-prohibitive due to the large numbers of individual reactions that must be performed. Microarray and sequencing can provide large amounts of data but are slow and expensive when only targeted panels are needed.




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