Illumination of a Critical Regulation Pathway of Protein Expression
Getting Started with MicroRNA Research

MicroRNAs (miRNAs) are a recently identified class of cellular RNAs that regulate protein expression at the translational level. The active, mature miRNAs are 17–24 base, single-stranded RNA molecules expressed in eukaryotic cells and are known to affect the translation or stability of target messenger RNAs. Each microRNA is believed to regulate multiple genes, with predictions that greater than one third of all human genes may be regulated by miRNA molecules [1].

Genes encoding approximately 230 miRNAs have already been identified in mammals [2]. Interestingly, more than 90% of the miRNAs are completely conserved among species, suggesting that these molecules are sensitive to small sequence changes and are potentially under extraordinary selection pressure. Here, we provide a general overview for analyzing miRNAs and describe some of the specialized tools that can help assess miRNA expression, function, and targets.

MicroRNAs (miRNAs) are highly conserved regulatory molecules expressed in eukaryotic cells. Data recently published in Cell suggest that the expression of certain genes can be more dependent on the levels of regulatory miRNAs than on the levels of messenger RNAs that encode the proteins [3]. MicroRNAs function through a mechanism similar to short interfering RNAs (siRNAs) in that both of these types of small, single-stranded RNA molecules target specific messenger RNA transcripts and prevent protein expression; however, miRNAs differ from siRNAs in that miRNAs are endogenous molecules encoded in the genomes of animals and plants (Figure 1). Given the importance of miRNAs, these biomolecules represent a tremendous opportunity to enhance our understanding of development, cell proliferation, differentiation, cell cycle, and disease (e.g., cancer and viral infections).

Figure 1. miRNA Processing Pathway. (1) miRNAs are expressed in the nucleus as parts of long primary miRNA transcripts (Pri-miRNA) that have 5’ caps and 3’ poly(A) tails. (2) The hairpin structure that likely forms around the miRNA sequence of the pri-miRNA acts as a signal for digestion by a double-stranded (ds) ribonuclease (Drosha) to produce the precursor miRNA (Pre-miRNA). (3) Exportin-5 mediates nuclear export of pre-miRNAs. (4) A cytoplasmic dsRNA nuclease (Dicer) cleaves the pre-miRNA leaving 1–4 nt 3' overhangs. The single-stranded mature miRNA associates with a complex that is similar, if not identical, to the RNA Induced Silencing Complex (RISC). (5) The miRNA/RISC complex represses protein translation by binding to sequences in the 3' untranslated region of specific mRNAs. The exact mechanism of translation repression is still undefined. *=mature miRNA sequence

The Research Questions
What miRNAs Exist?

Over the past several years, scientists have identified which miRNAs exist in each species. Most known miRNAs have been identified by random cloning and sequencing; investigators clone miRNAs by fractionating small RNA from a total RNA sample followed by cloning and sequencing these small RNA molecules (see MicroRNA Cloning Overview). There is an estimated 0.01% chance of uncovering a unique miRNA using this method--thus making discovery efforts very time-consuming and laborious. The miRNAs that remain to be characterized tend to be expressed in less commonly studied organisms and tissues. Recent studies have focused on bioinformatics, where algorithms predict miRNAs based on the presence of hairpins and other structures associated with the presence of miRNAs [4–8].

What Genes do miRNA Regulate?

While miRNA characterization is an active area of investigation, the importance of miRNAs lies in identifying the genes and biological pathways they regulate. One begins this process by examining the miRNA profiles in samples of interest (i.e., identifying which specific miRNAs are up- and down-regulated between samples). Specific miRNAs have already been linked to early stage development, cell differentiation, cell death, cancer, and regulation of viral infection, illustrating some of the critical roles that miRNAs play in cellular biology. However, of the 230 mammalian miRNAs, only 10 have been ascribed a function to date, leaving a lot of opportunity for discovery.

MicroRNA expression profiling involves extracting the small RNA fraction from the samples (such as normal and diseased tissues), and comparing the miRNA expression levels in each, for example, by array analysis. Isolation of miRNAs can be challenging due to the difficulties in purifiying these small RNAs away from larger nucleic acids as well as other small RNAs including tRNA, rRNA, and precursor miRNAs.

While most commercially available RNA isolation kits will not capture small RNAs (<200 nt), Ambion offers two kits that have been specifically optimized for the quantitative isolation of small RNAs (mirVanaT miRNA Isolation Kit) or small RNA and protein from the same sample (mirVana PARIST Kit).

Global miRNA expression profiling can be accomplished by microarray analysis; however, only mature miRNA should be used. To isolate mature miRNAs from precursor miRNA , the flashPAGET System, a miniature column electrophoresis system, can be used to quickly purify small nucleic acids without running traditional, time-consuming PAGE gels.

MicroRNA array profiling is performed by differentially labeling the resulting miRNA fractions from comparison samples with the mirVana miRNA Labeling Kit. The labeled miRNAs are then hybridized to an array that is spotted using the mirVana miRNA Probe Set, which contains probes to all known human, mouse, and rat miRNAs.

Following array analysis, microRNA expression should be confirmed by a secondary method, such as Northern blot analysis or the more sensitive solution hybridization assays (e.g., mirVana miRNA Detection Kit). Probes for Northern blot or solution hybridization analysis can be generated by using either the mirVana miRNA Probe and Marker Kit or the mirVana miRNA Probe Construction Kit.

What is the Function of Specific miRNAs?

MicroRNA functional analysis can be performed with protocols that are similar to those used to study standard genes: analysis of measurements from phenotypic responses or reporter assays. MicroRNA activity can be up-regulated to identify gain-of-function phenotypes and down-regulated or inhibited to identify loss-of-function phenotypes. Additionally, library screens of miRNA up- and down-regulation can be used to identify genes that are regulated by specific miRNAs as well as to identify cellular processes that are affected by specific miRNAs.

The Pre-miRT miRNA Precursor Molecules and Anti-miRT miRNA Inhibitors are used for analyzing miRNA function because they increase or decrease specific miRNA activity, respectively. Functional studies require quantitative, phenotypic assays (e.g., protein level, activity, or modification; protein or organelle transport; cell morphology or number; cell cycle status; membrane potential; calcium content; etc.) that can monitor changes in a specific biological process in response to up- or down-regulation of miRNA activity. Depending on pre-existing information about potential miRNA function (e.g., miRNA expression patterns or levels in various cell types), individual or a series of miRNAs can be targeted, and cells can be assayed for alterations in phenotype.

The pMIR-REPORTT miRNA Reporter Vector is a highly sensitive luciferase reporter vector that is useful for studying interactions between miRNA and target sites from mRNA transcripts, and can be used to measure relative miRNA activity levels. The cloning site in the 3' untranslated region (3' UTR) of the luciferase gene allows researchers to test potential miRNA target sites in cultured cells. In contrast, if miRNA target sites have already been characterized, treating cells with various compounds or artificially increasing or decreasing miRNA activity (e.g., through transfection of Pre-miR miRNA Precursor Molecules or Anti-miR miRNA Inhibitors) enables researchers to monitor miRNA activity.

The Complete miRNA Solution Provider™

Ambion has studied miRNAs for several years. During this time, we have developed a complete portfolio of technologies dedicated for the investigation of miRNAs that covers a complete experimental approach. See miRNA Experimental Overview for more information.

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