Determination of absolute expression profiles using multiplexed miRNA analysis

• Published in: PloS one Vol. 12; no. 7; p. e0180988
• Main Authors: Song, Yunke, Kilburn, Duncan, Song, Jee Hoon, Cheng, Yulan, Saeui, Christopher T, Cheung, Douglas G, Croce, Carlo M, Yarema, Kevin J, Meltzer, Stephen J, Liu, Kelvin J, Wang, Tza-Huei
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13.07.2017; Public Library of Science (PLoS)
• Subjects: Apoptosis; Assaying; Biology and life sciences; Biomarkers; Biomedical engineering; Biotechnology; Breast cancer; Cell cycle; Cell Line, Tumor; Cell lines; Copy number; Copy number variations; Deoxyribonucleic acid; DNA; DNA Copy Number Variations; DNA microarrays; Esophagus; Gene expression; Gene Expression Profiling - methods; Humans; Identification and classification; Leukemia; MCF-7 Cells; Mechanical engineering; Medicine; Medicine and Health Sciences; Methods; MicroRNA; MicroRNAs; MicroRNAs - genetics; miRNA; Multiplexing; Oligonucleotide Array Sequence Analysis - methods; Pancreas; Pancreatic cancer; Polymerase chain reaction; Properties; Research and analysis methods; Ribonucleic acid; RNA; Sizing; United States > US; Maryland; Baltimore Maryland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0180988

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.