microRNA - Streamlining the Workflow | Biowire Fall 2011


 

Biowire Fall 2011 — Screening — microRNA Target Identification

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Carol Kreader
Carol Kreader
Principle R&D Scientist, Sigma® Life Science

The importance of miRNA has recently come to the forefront of biomedical research. This issue of Biowire provides an overview of this exciting area of research and reviews Sigma’s solutions for the miRNA workflow.

The central dogma of molecular biology states that genetic information is transferred directionally from DNA that is transcribed to RNA, and this RNA is translated to protein. However, after completion of the human genome project, it was determined that the vast majority of DNA does not actually code for proteins. This non-coding sequence was first termed “junk DNA,” and, as the term implies, this DNA was thought to serve no purpose. Conventional wisdom began to change after publication of a 2004 Nature Reviews Genetics paper in which Mattick1 compared the ratio of non-coding to coding DNA across species, and observed that the more complex the species, the higher the ratio of non-coding to coding. Humans for example have a much higher ratio of non-coding to coding DNA compared to bacteria. Furthermore, in 2007, the ENCyclopedia Of DNA Elements (ENCODE) Consortium reported that most of the genome is transcribed as non-coding RNA2. These and subsequent studies clearly show that non-coding RNA is essential for organism complexity. The mechanism behind the complexity is believed to be the modulation of mRNA and protein levels by non-coding RNAs.

microRNAs were among the first non-coding RNA species to be described, and these in particular are now known to play an important role in gene regulation. miRNA-mediated regulation is essential for organism complexity and development, and aberrant expression of miRNAs and alterations in their binding sites have been implicated in numerous disease states, including cancer. miRNAs are short non-coding RNA molecules approximately 22 nt in size that function as post-transcriptional gene regulators by binding to complementary regions in their target mRNAs. The most common binding site is the 3' untranslated region (3'UTR) of the mRNA, however, miRNA targets in coding regions have also been reported. The mature miRNA, as part of the RNA Induced Silencing Complex (RISC), binds to its respective targets by recognizing regions of partial complementarity, and this typically results in inhibition of mRNA translation and/or mRNA destabilization and degradation. miRNAs do not require exact nucleotide matches to bind and regulate gene expression. Due to this imprecise nature of complementarity, a single miRNA can bind and inhibit multiple mRNAs. Furthermore, multiple miRNAs can bind and inhibit a single mRNA. This level of complexity has made it difficult for researchers to understand the role of miRNA in their system or disease of interest. There have been around 1,000 miRNAs identified in humans, and each miRNA has thousands of predicted putative miRNA binding sites in 3'UTRs. However, only a few hundred miRNA target genes have been confidently verified by experimentation.

One of the biggest challenges facing miRNA research is the identification of genes regulated by a specific miRNA. This challenge lies in the ability to correctly identify specific mRNA sequences that bind specific miRNAs and then verifying biological relevance. The most common method of target identification relies on computer algorithms to predict targets followed by validation with a luciferase reporter assay. This approach has proven to be very inefficient and ineffective as demonstrated by the high false positive and false negative rates of the in silico predictions, and the extremely low number of validated gene targets identified to date. To effectively identify genes regulated by miRNAs and other non-coding RNAs, researchers need assays that provide experimental ease-of-use and convenience, as well as reliable results. To address this need, Sigma® Life Science has partnered with King’s College London to develop a novel miRNA gene target identification system. The first product developed from this partnership is the MISSION® Target ID Library, which consists of human cDNA cloned downstream of a fusion protein, which allows for both positive and negative selection. This unique design allows for a global functional screen to isolate biologically relevant miRNA gene targets. In this issue of Biowire, we will describe in detail the development and use of the MISSION Target ID Library.

Sigma Life Science is committed to supplying researchers with the tools needed to better understand miRNA. With the addition of the Target ID Library to the MISSION RNAi product line, we now have products that facilitate three essential parts of the miRNA research workflow. These are outlined in Figure 1. The first step towards characterizing miRNA function is typically an assay to determine what role a miRNA plays in a specific disease or system. Typically, this is done by introducing a miRNA mimic into a cell line and analyzing the phenotype. Once the miRNA’s effect on phenotype is established, the next step is to identify the mRNA targets of that miRNA, and this is where the MISSION Target ID Library can be utilized. Finally, luciferase reporter-3'UTR constructs are an effective way to confirm gene targets found using the MISSION Target ID Library. We have recently introduced a lentiviral-based 3'UTR luciferase reporter system, the MISSION 3'UTR Lenti GoClones™. The MISSION 3'UTR Lenti GoClone constructs along with MISSION LightSwitch Reagents and MISSION miRNA mimics in combination provide miRNA researchers with an effective system to confirm gene targets for miRNA.

MISSION miRNA Workflow
Figure 1. The MISSION miRNA Workflow

Through development and collaboration with the research community, we have a continued commitment to provide innovative technologies in the field of miRNA and other non-coding RNA. The MISSION Target ID Library fills a large gap in the critical area of miRNA target identification. The MISSION miRNA mimics and the MISSION 3'UTR Lenti GoClones are also essential components in the miRNA research workflow. This is just the beginning, and with exclusive access to other exciting technologies, such as Zinc Finger Nucleases, we feel Sigma is in a unique position to make a significant impact on non-coding RNA research.

References

  1. Mattick JS. RNA regulation: a new genetics? Nature Reviews Genetics. 2004;5:316–323.
  2. Birney E, Stamatoyannopoulos JA, Dutta A, Guigo R, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007;447:799–816.

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