MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate the expression of thousands of genes in a broad range of organisms in both normal physiological contexts and in disease contexts. miRNA expression profiling is gaining popularity because miRNAs, as key regulators in gene expression networks, can influence many biological processes and also show promise as biomarkers for disease. Technological advances have spawned a multitude of platforms for miRNA profiling, and an understanding of the strengths and pitfalls of different approaches can aid in their effective use. Here, we review the major considerations for carrying out and interpreting results of miRNA-profiling studies. MicroRNAs (miRNAs) are small RNAs (∼22 nucleotides long) that post-transcriptionally regulate the expression of thousands of genes in a broad range of organisms.

miRNA expression profiling is useful for identifying miRNAs that are important in the regulation of a range of processes, including organismal development, tissue differentiation and disease pathology. miRNAs show promise as biomarkers for various diseases. miRNAs are more stable than mRNAs in many specimen types and are more readily measured than proteins. However, sample type, processing and RNA extraction methods can have a substantial impact on the results of miRNA profiling, and therefore quality and quantity assessment is recommended. Biogenesis of miRNAs occurs through multiple steps and includes the intermediate primary miRNA and precursor miRNA forms, as well as post-transcriptional nucleotide additions and deletions, leading to 'isomiRs'. Choice of platform and analysis in miRNA profiling should include consideration of the need to distinguish between different forms of miRNAs. Three main approaches are currently well established for miRNA profiling: quantitative reverse transcription PCR (qRT-PCR), hybridization-based methods (for example, DNA microarrays) and high-throughput sequencing (that is, RNA sequencing). The optimal choice of platform depends on the specific experimental goals.