Technical Article

rna-seqBiomarker identification is often associated with the diagnosis and evaluation of various diseases. Recently, the role of microRNA (miRNA) has been implicated in the development of diseases, particularly cancer. With the advent of next-generation RNA sequencing, the amount of data on miRNA has increased tremendously in the last decade, requiring new bioinformatics approaches for processing and storing new information. New strategies have been developed in mining these sequencing datasets to allow better understanding toward the actions of miRNAs. As a result, many databases have also been established to disseminate these findings.  Several databases are discussed below. (read more…)

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Researchers at the University of Oxford and the Scripps Research Institute recently published an article evaluating the use of several different RT-qPCR normalization strategies when profiling miRNA gene expression in mouse serum. In their research article entitled “Assessment of RT-qPCR Normalization Strategies for Accurate Quantification of Extracellular microRNAs in Murine Serum,” Roberts et. al. compare three miRNA expression normalization strategies: 1.) normalization of miRNA expression to the average Cq (Ct) of all measured miRNA genes, 2.) Normalization of gene expression to individual miRNAs such as miR-16, miR-223, or miR-31 (all miRNA genes previously identified as normalization controls in the literature), and 3.) Normalization to a ‘spike-in’ external control miRNA gene expressed in C. elegans (cel-miR-39).

Normalization strategies have been an increasingly important topic in miRNA expression studies. This is especially true when profiling miRNA expression in serum or tissues and when quantifying miRNA genes which can have considerable variability in their endogenous expression. The authors identified a panel of 124 serum miRNA genes present in all their samples between normal, dystrophic, and dystrophic-treated mice. Their results suggest that [click to continue…]

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The Role of MicroRNA ‘Sponges’

by Doug Dluzen on October 30, 2012

in Publications, Technical Article

The research and field of microRNA (miRNA) is relatively young in molecular biology. Researchers are only beginning to ascertain the essential functional impact that miRNA serve in tissue development and disease progression. For example, miRNA can act as both oncogenic ‘oncomirs’ or as tumor-suppressor genes in cancer biology. Amazingly, those same miRNA genes can also be critical to the function and cellular homeostasis of normal progenitor and mature cells. These findings illustrate the incredible promiscuity of miRNA functionality as well as their broad target specificity. Novel methods and techniques are needed not only to validate miRNA gene function; but also to identify pathway-related genes specific to both miRNA gene families as well as miRNA gene targets.

Dr. Phillip Sharp’s laboratory at MIT first proposed the concept of miRNA ‘sponges’ back in 20071. MiRNA sponges are plasmid constructs either transiently or stably transfected into mammalian cells containing multiple miR-binding sites for a chosen miRNA gene. The binding sites are located in tandem along an expressed transcript (see Figure 1). The ‘sponges’ are expressed via a strong promoter element and [click to continue…]

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microrna microarraysby Jeffrey M. Perkel

If PubMed is any guide, the microRNA field is smokin’ hot. Of the nearly 11,100 references that come up in a search for “microRNA,” nearly two-thirds (64%) were published since 2009.

It’s no wonder. Although microRNAs (miRNAs) may be small—they average 22 nucleotides in length—they carry a big stick, biologically speaking. “We can say with confidence that over 60% of human protein-coding genes are conserved targets of miRNAs,” wrote David Bartel and colleagues in 2009. [1]

To date, some 16,772 miRNAs have been discovered and logged in miRBase, including 1,424 in humans. The question for researchers probing these molecules’ biology is, which miRNAs are active under a given set of experimental conditions, and how does that pattern change in the dynamic cellular environment?

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Microarray-based analysis of cadmium-responsive microRNAs in rice (Oryza sativa).

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate specific target mRNAs at the post-transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptations to environmental stresses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to plants. To investigate the responsive functions of miRNAs under Cd stress, miRNA expression in Cd-stressed rice (Oryza sativa) was profiled using a microarray assay. A total of 19 Cd-responsive miRNAs were identified, of which six were further validated experimentally. Target genes were also predicted for these Cd-responsive miRNAs, which encoded transcription factors, and proteins associated with metabolic processes or stress responses. In addition, the mRNA levels of several targets were negatively correlated with the corresponding miRNAs under Cd stress. Promoter analysis showed that metal stress-responsive cis-elements tended to occur more frequently in the promoter regions of Cd-responsive miRNAs. These findings suggested that miRNAs played an important role in Cd tolerance in rice, and highlighted a novel molecular mechanism of heavy metal tolerance in plants.

Ding Y, Chen Z, Zhu C. (2011) Microarray-based analysis of cadmium-responsive microRNAs in rice (Oryza sativa). J Exp Bot [Epub ahead of print]. [article]

Microarray-based identification of tomato microRNAs and time course analysis of their response to Cucumber mosaic virus infection.

Abstract: A large number of plant microRNAs (miRNAs) are now documented in the miRBase, among which only 30 are for Solanum lycopersicum (tomato). Clearly, there is a far-reaching need to identify and profile the expression of miRNAs in this important crop under various physiological and pathological conditions. In this study, we used an in situ synthesized custom microarray of plant miRNAs to examine the expression and temporal presence of miRNAs in the leaves of tomato plants infected with Cucumber mosaic virus (CMV). Following computational sequence homology search and hairpin structure prediction, we identified three novel tomato miRNA precursor genes. Our results also show that, in accordance with the phenotype of the developing leaves, the tomato miRNAs are differentially expressed at different stages of plant development and that CMV infection can induce or suppress the expression of miRNAs as well as up-regulate some star miRNAs (miRNA*s) which are normally present at much lower levels. The results indicate that developmental anomalies elicited by virus infection may be caused by more complex biological processes.

Lang QL, Zhou XC, Zhang XL, Drabek R, Zuo ZX, Ren YL, Li TB, Chen JS, Gao XL. (2011) Microarray-based identification of tomato microRNAs and time course analysis of their response to Cucumber mosaic virus infection. J Zhejiang Univ Sci B. 12(2),116-25. [abstract]

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RNA-Seq Contributing to microRNA Discoveries

February 14, 2011

The advent of next-generation sequencing technologies provides new opportunities to profile transcriptomes and microRNA targetomes at base-wise resolution.  A couple of recent posts on the RNA-Seq Blog highlight some of the recent discoveries in the microRNA field made possible by RNA-Seq technologies. microRNA Discovery by RNA-Seq microRNA Isoform Target Prediction by RNA-Seq Incoming search terms […]

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microRNA Biomarkers in Cerebrospinal Fluid

February 1, 2011

The identification of microRNA biomarkers in the cerebrospinal fluid may become an important method for the diagnosis of primary central nervous system disorders. Researchers have identified and quantified microRNAs in cerebrospinal fluid samples taken from patients with primary central nervous system lymphomas and as well as patients with a variety of neurologic disorders, including inflammatory […]

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microRNAs a critical factor in cerebral, renal, and cardiovascular ischemia

January 11, 2011

MicroRNAs are approximately 22 nucleotide long, noncoding RNAs that extensively mediate post-transcriptional gene expression.  They control cellular function by either degrading mRNAs or arresting their translation. MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis as well as (patho)physiologic conditions such as tumor progression/regression and cholesterol/glucose homeostasis. […]

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microRNAs as Biomarkers in Plasma

October 20, 2010

MicroRNAs (miRNAs) are small non-coding RNAs found in eukaryotic organisms that regulate gene expression. Dismissed as “junk” until about a decade ago, it is now widely accepted that they play an important functional role in a wide array of cellular processes. MiRNAs regulate gene expression by targeting individual or multiple messenger RNAs (mRNAs). Perfect or […]

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microRNAs Associated with Cancer

October 12, 2010

Found this concise table of human microRNAs relating to cancer in a recent review article1.  Anyone care to add to this based on their own research? Cancer Type Human microRNA Potential Function Breast Cancer miR-21, miR-125b; miR126, miR-206, miR-335 OncomiR; Metastasis Suppressor Colon Cancer miR-21; miR-34a OncomiR; Tumor Suppressor Lung Cancer miR-21, miR17-92 cluster, miR-106b/93/25 […]

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