MicroRNAs (miRNAs) are a class of small (~19-23 nucleotides), non-protein coding RNA molecules found in a broad range of plants, viruses and mammals. miRNAs are initially transcribed in the cell nucleus as long precursors (pri-miRNA) that are enzymatically processed into ~70 nucleotide stem-loop structures (pre-miRNA), which are then exported into the cell cytoplasm. A second enzymatic processing step creates ~22 nucleotide long mature miRNAs. These mature miRNAs can regulate gene expression following incorporation into an active RNA-induced silencing complex (RISC) where they interact with complementary sites on messenger RNA molecules resulting in translational repression and sometimes degradation of the target messenger RNA. While identification of the characteristic stem-loop structure of pre-miRNA is important for miRNA classification,[1
] it is the expression level of the mature form that is of most interest to researchers investigating the biological function of miRNAs. There are a number of good reviews available on the mechanism of gene regulation and silencing by miRNAs.[2
Although the first published description of a miRNA appeared more than a decade ago,[7
] the importance of miRNA in gene regulation and cell function is just beginning to be understood. Studies in model organisms have shown miRNAs are involved in the regulation of many critical biological processes such as development, differentiation, metabolism and immunological response.[8
] Recently, several research groups have established links between alterations in the expression levels of miRNAs and the initiation and development of human cancers.[11
] Comparative analyses between various malignant and normal tissue samples revealed characteristic patterns, where some miRNAs are overexpressed and others strongly repressed, that depended on the cancer type, disease stage and response to treatment.[12
] Also, miRNA expression levels have been shown to play a controlling role in tumor growth rates suggesting possible new strategies for therapeutic treatment.[13
] A key approach emerging in the investigation of the cellular roles of miRNAs is to profile the mature miRNA expression levels in specific tissue types at various developmental or disease stages.
For this reason, considerable effort has been devoted to the development of new methods for high throughput, multiplexed miRNA gene expression analysis. Currently, over 4500 miRNA sequences are listed in the miRNA Registry,[14
] including 475 human miRNAs. The total number of miRNAs in the human genome is not yet known, but estimates based on computational analyses range up to 1000.[2
] Recently, Tuschl and colleagues published one of the most comprehensive survey of mammalian miRNA expression patterns to date where they used a variety of experimental and computational methods to analyze miRNA expression profiles from 26 different organ systems and cell types of humans and rodents.[17
] A timeline for miRNA discovery and detection is shown in . The earliest attempts at systematically profiling miRNA expression were performed using a large number of single miRNA detection experiments such as northern blotting.[18
] Although laborious and sample intensive, multiplexed northern blotting continues to be widely used as the standard method against which data from newer, more sensitive detection techniques are validated. Other multiplexed single miRNA approaches such as a modified invader assay[19
] and quantitative RT-PCR of precursor miRNAs[20
] or mature miRNAs[21
] are very sensitive and require low amounts of starting material. The successful application of real-time PCR is the result of innovative approaches towards the design of RT primers with high specificity towards individual mature miRNA species[22
]; potentially, multiple primers could be combined in a single pool that would enable much higher-throughput profiling than currently possible with individual sample analyses.
Figure 1 Timeline for microRNA discovery and detection. Initially, expression analysis was performed using northern blotting. Cloning and sequencing methods were later used to discover hundreds more miRNAs. The improved understanding of miRNA properties has enabled (more ...)
In recent years, these multiplexed single miRNA methods have been supplanted by the use of oligonucleotide microarray-based detection platforms as the most efficient approach to miRNA high-throughput profiling.[23
] Complementary DNA (cDNA) microarrays for the multiplexed detection of messenger RNA molecules have proven to be extremely valuable for the study of gene expression in biological samples[33
]; in a similar fashion, the population of known miRNA molecules should in principle be detected and characterized by creating DNA microarrays with hundreds to thousands of complementary probe sequences immobilized on a single chip surface. However, the short sequence length of miRNAs (19-23mers) means that the methods used to detect messenger RNA with oligonucleotide microarrays cannot be used. In addition, direct PCR amplification of mature miRNAs cannot be used, making the ultrasensitive detection of miRNA at femtomolar concentrations particularly difficult. In this Minireview, we focus on the latest developments that overcome the various difficulties in the application of microarray-based detection platforms for the profiling of miRNA expression in biological samples.