MicroRNAs in disease discovery

Further understanding of gene expression could lead to faster diagnosis and lower incidence of disease. One company’s microRNA mimicking system is helping to expand the market for genetic regulation.

MicroRNAs (miRNAs) are emerging not only as key regulators of gene expression in fundamental cellular physiology but also potential biomarkers in the characterisation of normal biological processes as well as the diagnosis and prognosis of disease states. Animal miRNAs have been shown to regulate many critical aspects of fundamental cellular physiology, including developmental transitioning and timing, proliferation, apoptosis, differentiation and metabolism. Given their importance in the maintenance of basic cellular processes, it stands to reason that deregulation of miRNA activity can lead to disease. Analysis and alteration of miRNA activity presents significant opportunities in the characterisation, diagnosis, prognosis and, ultimately, the treatment of human disease.

Global expression profiling
Standard genome-wide techniques such as microarray-based expression profiling have been expanded to encompass miRNAs. Characterising miRNA expression in a given disease state or therapeutic regimen yields an easily manageable, unique molecular fingerprint. This has intrinsic diagnostic and prognostic value and can be applied to better understand the biologic process of the disease and a drug’s mechanism of action in the treatment of that disease.

The Thermo Scientific Dharmacon miRNA expression profiling system is a sensitive platform developed to accurately detect expression levels from nanogram quantities of total RNA. A synthetic miRNA reference probe standard allows precise and reproducible data normalisation. It consists of an equimolar mixture of synthetic dye-labelled miRNA molecules that is hybridised together with the labelled biological sample to the microarray. This sample is used as a common reference to experimentally control for any differences that may occur during hybridisation and is used as a basis for normalising the expression profiling data.

The resulting normalised relative intensity data format is comparable to output from single-channel expression profiling platforms and can be analysed in a similar manner. Unlike dual-channel experiments, the normalised relative intensity data is readily comparable between experiments and can be used to analyse data generated between experiments separated in time.

Specific modulation
MiRNAs have been shown to regulate gene expression through translational attenuation and messenger RNA (mRNA) degradation. These small non-coding RNAs typically target multiple genes simultaneously, inducing subtle but reproducible shifts in target gene expression. Synthetic miRNA mimics and inhibitors designed for potency and specificity can be valuable tools for probing the function of target genes. Major challenges include the confirmation of subtle phenotypic changes associated with over-expression and inhibition as well as identification of gene targets.

An emerging approach is the identification of miRNAs by screening with synthetic reagents that mimic or inhibit function to demonstrate a specific phenotypic effect. Endogenous miRNAs are transcribed as stem-loop structures and subsequently processed by several enzymes, including the RNA-induced silencing complex (RISC), into functional, mature miRNAs with one or two mature (targeting) strands. Thermo Scientific Dharmacon miRIDIAN miRNA Mimics are synthetic duplexes loaded into RISC and then able to regulate mRNA to target sites in the 3’ UTR of a particular transcript. By introducing these molecular mimics into a cell type of interest, the researcher can enhance or supplement endogenous miRNA activity representing a gain-of-function assay.

Conversely, the addition of miRNA inhibitors into a biological system results in a loss-of-function assay with a predicted decrease in endogenous miRNA function. The mechanism of inhibition is likely mediated by binding of the inhibitor to mature miRNA, preventing binding of the miRNA to its endogenous targets. Inhibitor potency can be improved by enhancing binding through chemical modification and/ or by incorporating additional attributes, such as structure, into the molecule. The newest generation of inhibitors, miRIDIAN miRNA Hairpin Inhibitors, incorporate terminal stem-loop structures and chemical modifications to enhance potency and extend inhibitory activity. MiRNA expression profiling coupled with phenotypic screening permits the identification and validation of microRNA function in the pathology of disease and the profiling of responses to therapeutic intervention. These neobiomarkers are poised to create a bridge between preclinical observations and clinical efficacy and safety

Company profile

Thermo Fisher Scientific provides valuable tools for the analysis and modulation of mRNAs to generate data that has potential diagnostic and prognostic value for evaluating drug safety and characterising human disease.

For further information, visit: www.thermofisher.com.