microRNA Profiling

An Overview of Current Technologies and Applications

Sinead M. Smith*; David W. Murray†    * Department of Clinical Medicine and School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland
† Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland

1 Introduction

Recent insight into the transcriptional landscape of mammalian genomes gained from high-throughput next-generation sequencing (NGS) technologies has revealed that although 80% of the genome is transcribed, less than 2% is subsequently translated into protein, resulting in the generation of a large number of ncRNA transcripts (1–5). Although originally considered to be nonfunctional, recent data have highlighted that noncoding RNAs (ncRNAs) play key roles in gene regulation by influencing transcription and translation of target genes, thus regulating a diverse range of biological processes under normal physiological conditions and in disease settings (6). Broadly speaking, ncRNAs may be subdivided according to their size. First, long ncRNAs (lncRNAs) ranging in size from a few hundred nucleotides (nt) to multiple kilobases in length represent the largest class of ncRNAs and account for much of the transcribed genome (6). In contrast to lncRNAs, small ncRNAs are less than 300 nt in length and include microRNAs (miRNAs), piwi-interacting RNAs, small-interfering RNAs, and small nuclear RNAs (snRNAs) among others (7). The miRNAs (18–25 nt) are the most widely studied family of the small ncRNAs. Since their discovery in 1993 (8,9), the diversity and significance of this class of regulatory molecule has become increasingly appreciated. miRNAs posttranscriptionally decrease the expression of thousands of target genes by binding to specific messenger RNA (mRNA) targets and promoting their degradation and/or inhibiting their translation (10–12). Although a relatively limited number of miRNAs (approximately 1000) have been identified in humans compared with the number of mRNAs and proteins (approximately 30,000), a single miRNA may regulate hundreds of mRNAs and thus has the potential to greatly impact gene-expression networks (10).

The importance of miRNAs in biological processes is demonstrated by their high levels of evolutionary conservation (13). Accumulating evidence supports a role for miRNAs in normal cellular physiology, where they act as key regulators of development (14), differentiation (15,16), cell proliferation, and apoptosis (17,18). For example, both specific miRNAs and proteins involved in miRNA processing play fundamental roles in the development and function of B- and T-cells within the immune system (19–23). Additionally, miRNAs coordinate cellular responses in innate immune cells (24) and play key roles in the regulation of host–pathogen interactions during infection (25). miRNA expression is also regulated during inflammation (25–27) and as a consequence perturbed miRNA expression is associated with a number of autoimmune diseases, including multiple sclerosis (28), rheumatoid arthritis (29,30), and systemic lupus erythematosus (31,32). Furthermore, expression profiling has implicated miRNAs in numerous cancers, including B-cell chronic lymphocytic leukemia (33) and cancer of the breast (34–37), colon (35,38,39), liver (40,41), and lung (35,42–47), among others. miRNAs function as both tumor suppressors and oncogenes (48,49). Given the key roles of miRNAs in normal cellular homeostasis and their dysregulation under disease conditions, investigation of miRNA expression profiles and regulation of their mRNA targets is essential for a complete understanding of the cell signaling mechanisms that mediate cell function in health and disease, with the potential to identify new therapeutic agents or drug targets. Moreover, as miRNAs are stable in a variety of clinical specimens, including formalin-fixed paraffin-embedded (FFPE) tissue, blood, and urine, there is substantial interest in their development as biomarkers for diagnostic applications. Indeed, profiling of circulating miRNAs has already shown promise for detection of cancers (36,44,45,50–53).

2 miRNA Biogenesis and Nomenclature

An understanding of the multistep processes involved in miRNA processing and biogenesis is required for the design and application of analytical techniques for miRNA detection and quantitation. Primary miRNA (pri-miRNA) transcripts are either transcribed by RNA polymerase II from independent genes or represent introns of protein-coding genes (54–56). A pri-miRNA transcript can contain a single miRNA or multiple miRNAs that are processed from the same transcript (25). The pri-miRNA folds into a hairpin structure, which acts as a substrate for cleavage by the endonuclease Drosha resulting in an approximately 70–100 nt long precursor miRNA (pre-miRNA) (Figure 1). Following the Drosha cleavage, the pre-miRNA is exported to the cytoplasm by Exportin, where it is further processed by the endonuclease Dicer to produce an miRNA duplex comprised of miRNA strands derived from the 5′ and 3′ regions of the precursor duplex (7,10,55). One strand of this duplex, representing a mature miRNA, is incorporated into the RNA-induced silencing complex (RISC), while the other passenger strand is usually degraded. As part of the RISC, miRNAs base pair with complete or partial complementarity to sequences in the 3′ untranslated region (3′-UTR) of target mRNAs and induce mRNA translational repression or instability by deadenylation and degradation (55).

Different mature miRNA species can be produced from a single pre-miRNA molecule, as distinct miRNAs are generated from the 3′ and 5′ arms of the pre-miRNA duplex. In addition, a given mature miRNA may comprise a distribution of sizes centered around 22 nt rather than a discrete length. The variation in mature miRNA length is due to 3′ or 5′ end...