MicroRNAs (miRNAs) constitute a critically important class of non-translated, small RNAs which post-transcriptionally regulate gene expression via one of multiple mechanisms.. [1
] First reported in 1993 as a curious anomaly in Caenorhabditis elegans
] thousands of miRNAs have now been identified and shown to play key roles in many transformative biological processes, including developmental timing, [3
] stem cell differentiation, [6
] and disease development. [9
] Although the complete functional role that miRNAs play still remains to be fully elucidated, their conservation throughout Archaea, [11
] bacteria, [12
] plants, [13
] and animals[14
] indicate their importance as key regulatory control elements during both normal and transformative biological processes. In contrast to small interfering RNAs (siRNAs), [15
] miRNAs are endogenously encoded into the genome and are initially transcribed as long primary transcripts (≥1 kb; pri-miRNAs), which are then enzymatically processed in the nucleus by Drosha into ~70 nt stem loop structures (pre-miRNAs). Pre-miRNAs are exported into the cytoplasm and processed by the enzyme Dicer into the mature 19–24 nt duplexes.
As opposed to siRNAs, which operate almost exclusively via mRNA cleavage at regions having perfect sequence complementarity, miRNAs can modulate gene expression via one of three distinct mechanisms and do not necessarily require perfect base pairing to act upon a target. [1
] In the cytoplasm, the single strands form the mature miRNA duplexes are incorporated into the RNA-induced silencing complex (RISC). Guided by the miRNA, the RISC complex can then act on mRNAs through one of three distinct mechanisms: 1) cleavage of the targeted mRNA, a mechanism commonly observed in plants that often requires perfect complementarity between miRNA and mRNA, 2) translational repression, whereby miRNA/RISCs bind to 3 untranslated regions of mRNAs preventing translation by the ribosome, and 3) the recently discovered enhancement of translation, in which a miRNA binds to the 5′-terminal oligopyrimidine tract (5′-TOP) and relaxes a cis-element in the 5′ UTR that inhibits translation. [16
There are over 15,000 mature miRNA sequences listed in the recently released miRBase 15.0 database, with ~1000 identified as human miRNAs. [17
] Through one or more of the aforementioned mechanisms, each miRNA can potentially regulate the expression of multiple mRNAs, meaning that downstream production of many gene products, ultimately proteins, can be tremendously influenced by alterations in the expression of a single miRNA. [18
] In fact, it is known that a majority of human mRNAs are regulated by one (or more) miRNAs. [19
] Furthermore, it has recently been experimentally demonstrated that multiple miRNAs, many of which are expressed as clusters that are encoded in close genomic proximity to one another, can target the same mRNA, [20
] adding further complexity to the mechanisms through which miRNAs regulate gene expression.
Given the prominent role that miRNAs play in “normal” gene expression and organismal function, it is not surprising that the aberrant expression of miRNAs can lead to a wide range of human diseases and disorders, including: cancer, [21
] neurodegenerative diseases, [23
] diabetes, [25
] heart diseases, [26
] kidney diseases, [27
] liver diseases, [29
] and altered immune system function, [30
] amongst others. In addition to contributing to the underlying cause of a particular disease, miRNAs can also represent potential therapeutic targets[32
] and diagnostic biomarkers. [35
] Particularly exciting are the discovery of circulating miRNAs, which are promising biomarker candidates since they can be detected from readily attainable blood samples. [36
Almost entirely due to their short size, the analysis of miRNAs is considerably more difficult than it is for much longer mRNAs. In particular, the small size of miRNAs greatly complicates the use of standard molecular biology methods based upon the polymerase chain reaction (PCR), as detailed below. Furthermore, the short size also makes hybridization-based assays difficult as the melting temperature and binding dynamics of complementary probes toward their target miRNAs vary significantly with the identity of the target miRNA. Furthermore, experimental parameters, such as the buffer composition, the hybridization temperature, and incubation time all can contribute to significant assay-to-assay variation. [39
So, what are desirable attributes for existing and emerging miRNA analysis methods? Clearly the most appropriate technique for a given measurement challenge varies tremendously based upon the application and setting. For example, in an academic laboratory setting well-established techniques that rely upon the tools of traditional molecular biology might find favor, whereas emerging micro- or nanotechnology-based methods might eventually be most well-suited for point-of-care diagnostic applications. Two other important considerations when selecting an existing or designing a new method for miRNA analysis include dynamic range and multiplexing capability. The expression level of miRNAs, as determined via intracellular copy number, can vary from sequence to sequence by up to a factor of 105 within a single sample. Furthermore, the recent discoveries of multiple miRNAs targeting a single mRNA and regulated expression amongst entire families of miRNAs provide motivation for global miRNA analyses, which will require methods wherein multiple miRNAs, and perhaps the entire “miRNA-ome”, is simultaneously detected in parallel in order to fully elucidate the important and complex function of these tiny regulators.
On account of the critical biological role that miRNAs play in biological function and the diverse range of applications in which miRNA analysis is of value, significant effort has been invested over the past decade to develop new detection methods. In this critical review we highlight a selection of existing and emerging tools for miRNA analysis, with a particular emphasis on the current state-of-the-art and important developments in this fast moving field, as reported in the primary literature in the past four years.