Robust global microRNA expression profiling using next-generation sequencing technologies.
miRNAs are a class of regulatory molecules involved in a wide range of cellular functions, including growth, development and apoptosis. Given their widespread roles in biological processes, understanding their patterns of expression in normal and diseased states will provide insights into the consequences of aberrant expression. As such, global miRNA expression profiling of human malignancies is gaining popularity in both basic and clinically driven research. However, to date, the majority of such analyses have used microarrays and quantitative real-time PCR. With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal many more options. To make effective use of these different platforms, the strengths and pitfalls of several miRNA profiling technologies were assessed, including a microarray platform, NGS technologies and the NanoString nCounter System. Overall, NGS had the greatest detection sensitivity, largest dynamic range of detection and highest accuracy in differential expression analysis when compared with gold-standard quantitative real-time PCR. Its technical reproducibility was high, with intrasample correlations of at least 0.95 in all cases. Furthermore, miRNA analysis of formalin-fixed, paraffin-embedded (FFPE) tissue was also evaluated. Expression profiles between paired frozen and FFPE samples were similar, with Spearman's ρ>0.93. These results show the superior sensitivity, accuracy and robustness of NGS for the comprehensive profiling of miRNAs in both frozen and FFPE tissues.
1] Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada [2] Princess Margaret Cancer Centre, University Health Networks, Toronto, ON, Canada [3] Genome Technologies, Ontario Institute for Cancer Research, Toronto, ON, Canada.
Shirley
S
Tam
Informatics and Bio-Computing, Ontario Institute for Cancer Research, Toronto, ON, Canada.
Richard
R
de Borja
1] Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada [2] Princess Margaret Cancer Centre, University Health Networks, Toronto, ON, Canada [3] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
Ming-Sound
MS
Tsao
1] Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada [2] Genome Technologies, Ontario Institute for Cancer Research, Toronto, ON, Canada [3] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
John D
JD
McPherson
D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D064593:Heterografts; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006652:Histological Techniques; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D035683:MicroRNAs / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods
D035683:MicroRNAs; D012334:RNA, Neoplasm