High-throughput approaches for microRNA expression analysis.

Profiling microRNA (miRNA) expression is of widespread interest due to their critical roles in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Profiling can be achieved via three major methods: amplification-based (real-time quantitative PCR, qRT-PCR), hybridization-based (microarrays), and sequencing-based (next-generation sequencing (NGS)) technologies. The gold standard is qRT-PCR and serves as a platform for single reverse PCR amplification experiments and for a large number of miRNAs in parallel, both by multiplexing and plate based arrays. Currently, qRT-PCR is used for the validation of miRNA profiling results from other platforms. Hybridization based miRNA profiling by microarrays has become a widely used method especially for biomarker and therapeutic target identification. The data obtained from microarrays also enables functional prediction of miRNAs by correlating miRNA expression patterns to corresponding mRNA and protein profiles. Additionally, miRNA profiling strategies based on deep sequencing allow both the identification of novel miRNAs and relative quantification of miRNAs. Each miRNA profiling strategy has specific strengths and challenges that have to be considered depending on the nature of the research context.In this chapter the high-throughput approaches that can be applied to microRNA profiling are discussed starting from small-scale qRT-PCR technology to a wider one, NGS.