MicroRNAs:
How These Small Fragments of RNA are Having a Large Impact

MicroRNA (miRNA) were first identified in 1993 during the study of C. elegans development but have since been found in nearly every species of plant and animal. The role in embryogenesis of miRNA was just the tip of the iceberg, as these powerful regulators of gene expression have been implicated in such diverse areas as the immune response, neural development, DNA repair, apoptosis, oxidative stress response and others. It is the goal of this summary to give an overview of how miRNA, a new level of genetic regulation, are important to both basic as well as applied research in the life sciences.

What is miRNA?

Micro RNA are tiny, typically 22 nucleotides in length, and are produced from larger precursor RNA transcripts (about 70 nucleotides) by enzymes in the Argonaute family and the RNase III, Dicer. The miRNA is then incorporated into the RNA-induced silencing complex (RISC) which suppresses the translation or enhances the degradation of target messenger RNA (mRNA) molecules, thereby reducing the synthesis of the encoded protein. The miRNA acts by binding to the 3'-untranslated region (3'-UTR) of the mRNA, making it possible for a single miRNA to control multiple genes with the same sequence in this region of the mRNA.

In vertebrates, the RISC complex is guided to its mRNA target by the miRNA strand, which typically base pairs imperfectly to its target in the 3'-UTR, signaling the target for translational repression through unknown mechanisms. More than 1000 miRNA sequences for humans have been identified and cataloged in the microRNA database miRBase (http://microrna.sanger.ac.uk/sequences/index.shtml), and each miRNA is proposed to have hundreds of mRNA targets due to their imperfect base pairing. Therefore, the bioinformatics prediction that 30% of human genes are regulated by miRNA can be seen as a reasonable assumption.

Relevance of miRNA to Human Biology

Before the discovery of miRNA, it had been known that a large part of the genome is not translated into proteins. This so called "junk" DNA was thought to be evolution's debris with no function. We now realize that a portion of this non-coding DNA is highly relevant in the regulation of gene expression. While only about 500 human miRNA sequences have been identified so far, genomic computational analysis indicates that as many as 50,000 miRNA may exist in the human genome, and each may have multiple targets based on similar sequences in the 3'-UTR of mRNA.

The importance of the miRNA regulatory pathways is underscored by the impressive list of diseases which have recently been found to be associated with abnormal miRNA expression.

Cancer: miRNAs have been found to be down-regulated in a number of tumors, and in some cases, the reintroduction of these miRNA has been shown to impair the viability of cancer cells. The value of miRNA profiles in tumor diagnostics is well established. For instance, strong up and down regulations of miRNA sequences have been shown in primary breast tumors, and these markers may aid in the development of tests for drug-resistance and for treatment selection. Underlining the important role miRNA plays in oncology is the formation of several new companies which seek to expand development of miRNA-based therapeutics.

Age Related Diseases: Evidence is accumulating that many age-related diseases are associated with a decreased signaling control that occurs in mid-life. The miRNA controlling such systems as the cell cycle, DNA repair, oxidative stress responses, and apoptosis have been shown to become abnormally expressed in midlife. It is highly likely that continued research will reveal important associations with the aging process, and may lead to therapeutics that can improve the quality of life.

Heart Disease: Two heart specific miRNA sequences were deleted in mouse models resulting in abnormal heart development in a large proportion of the offspring. While these lethal effects were expected, other studies show a more subtle role for miRNA in the heart. When miR-208 was eliminated, the mice appeared normal. Only when their hearts were stressed, did defective responses show up. These results show that comprehensive miRNA studies may be valuable in the diagnosis of heart disease.

Neurological Diseases: Numerous reports have demonstrated the role of miRNA in neural development. Evidence for a role in Parkinson's disease comes from animal model studies showing that loss of miRNA may be involved in the development and progression of the disease. In cell culture experiments, transfer of small RNA fragments partially preserved miRNA-deficient nerve cells. While these results and others point to an important role for miRNA in neurodegenerative disorders, much more work is needed to delineate the exact role of miRNA in this important area.

Immune Function Disorders: miRNA deletion studies have revealed a central role for them in the regulation of the immune response. The deletion of miRNA-155 impaired T and B cell differentiation in germinal centers and greatly decreased antibody and cytokine production. Two additional studies deleting miRNA-181 and 223 were found to control T cell response and granulocyte production, respectively. As more roles for miRNA in the immune response are found, the list of immune function disorders with a miRNA component is certain to expand.

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Detecting every miRNA across the entire genome in a specific and sensitive way is a very challenging technology task. Many miRNA family members and otherwise distinct miRNA species have very similar sequences. Moreover, other RNA species such as snRNA, tRNA, mRNA, and rRNA can cause non-specific amplification, making the specific analysis of mature miRNA even more problematic. QIAGEN SABiosciences' proprietary miRNA technologies, including a miRNA isolation technique, primer design methodology, and assay formulation for universal reverse transcription specific to miRNA, address these challenges. QIAGEN's complete miRNA PCR System includes:

• miScript miRNA PCR Arrays and Assays
• miScript II Reverse Transcription Kits
• miScript SYBR® Green PCR Kits
• miRNeasy and PAXgene miRNA Isolation Kits

miScript miRNA PCR Arrays and Assays dramatically improve the specificity for and discrimination of closely related miRNA sequences and distinguish mature miRNA from other RNA species. Our miRNA PCR Arrays include built-in control elements to insure the quality of your experimental data. The free data analysis software takes your raw threshold cycle data and automatically generates figures and tables ready for publication. With the miScript miRNA PCR Assays and Arrays, you can expect:

• Sensitivity: As little as 125 ng total RNA needed for a Pathway Array
• Multi-Sequence Flexibility: Analyze one to 372 sequences simultaneously
• Simplicity: As easy as a real-time PCR experiment

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Published References