microRNA REVIEWs  (6)
microRNA  (miRNA)   &   quantitative real-time RT-PCR (1)
microRNA  (miRNA)   &   quantitative real-time RT-PCR (2)
microRNA  (miRNA)   &   quantitative real-time RT-PCR (3)
microRNA  (miRNA)   &   quantitative real-time RT-PCR (4)
microRNA  (miRNA)   &   quantitative real-time RT-PCR (5)
microRNA normalisation (7)
mirtrons  (8)
latest microRNA papers (9)  ... NEW

RNA interference (RNAi)        small inhibiting RNA  (siRNA)       small activating RNA  (saRNA)

Tiny molecules called microRNAs are tearing apart traditional ideas about the animal family tree

microRNA Reviews
microRNA normalisation ... UPDATED !

REWRITING EVOLUTION  -  Tiny molecules called microRNAs are tearing apart traditional ideas about the animal family tree

Kevin Peterson grabs a pen and starts to scribble an evolutionary tree on the paper tablecloth of a bar in Hanover, New Hampshire. Drawing upside down to make it easier for me to see, he maps out the standard phylogenetic tale for placental mammals. First, Peterson scratches a line leading to elephants, which branched away from the rest of the placentals around 90 million years ago. Then came dogs, followed by primates (including humans) and finally rodents — all within a frenetic 20 million years. This family tree is backed up by reams of genomic and morphological data, and is well accepted by the palaeontological community. Yet, says Peterson, the tree is all wrong.


MicroRNAs and other non-coding RNAs as targets for anticancer drug development.
Ling H, Fabbri M, Calin GA.
Nat Rev Drug Discov. 2013 12(11): 847-865

The first cancer-targeted microRNA (miRNA) drug - MRX34, a liposome-based miR-34 mimic - entered Phase I clinical trials in patients with advanced hepatocellular carcinoma in April 2013, and miRNA therapeutics are attracting special attention from both academia and biotechnology companies. Although miRNAs are the most studied non-coding RNAs (ncRNAs) to date, the importance of long non-coding RNAs (lncRNAs) is increasingly being recognized. Here, we summarize the roles of miRNAs and lncRNAs in cancer, with a focus on the recently identified novel mechanisms of action, and discuss the current strategies in designing ncRNA-targeting therapeutics, as well as the associated challenges.

MicroRNA profiling: approaches and considerations.
Pritchard CC, Cheng HH, Tewari M.
Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
Nat Rev Genet. 2012 Apr 18;13(5): 358-369

MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate the expression of thousands of genes in a broad range of organisms in both normal physiological contexts and in disease contexts. miRNA expression profiling is gaining popularity because miRNAs, as key regulators in gene expression networks, can influence many biological processes and also show promise as biomarkers for disease. Technological advances have spawned a multitude of platforms for miRNA profiling, and an understanding of the strengths and pitfalls of different approaches can aid in their effective use. Here, we review the major considerations for carrying out and interpreting results of miRNA-profiling studies.
Ribo-gnome: The Big World of Small RNAs
Phillip D. Zamore and Benjamin Haley

Small RNA guides—microRNAs, small interfering RNAs, and repeat-associated small interfering RNAs, 21 to 30 nucleotides in length—shape diverse cellular pathways, from chromosome architecture to stem cell maintenance. Fifteen years after the discovery of RNA silencing, we are only just beginning to understand the depth and complexity of how these RNAs regulate gene expression and to consider their role in shaping the evolutionary history of higher eukaryotes.

microPrimer: the biogenesis and function of microRNA
Du T, Zamore PD.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Development. 2005 Nov;132(21): 4645-4652

Discovered in nematodes in 1993, microRNAs (miRNAs) are non-coding RNAs that are related to small interfering RNAs (siRNAs), the small RNAs that guide RNA interference (RNAi). miRNAs sculpt gene expression profiles during plant and animal development. In fact, miRNAs may regulate as many as one-third of human genes. miRNAs are found only in plants and animals, and in the viruses that infect them. miRNAs function very much like siRNAs, but these two types of small RNAs can be distinguished by their distinct pathways for maturation and by the logic by which they regulate gene expression

The widespread regulation of microRNA biogenesis, function and decay.
Krol J, Loedige I, Filipowicz W.
Friedrich Miescher Institute for Biomedical Research, 4002 Basel, Switzerland.
Nat Rev Genet. 2010 11(9):597-610

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that are approximately 21 nucleotides in length and control many developmental and cellular processes in eukaryotic organisms. Research during the past decade has identified major factors participating in miRNA biogenesis and has established basic principles of miRNA function. More recently, it has become apparent that miRNA regulators themselves are subject to sophisticated control. Many reports over the past few years have reported the regulation of miRNA metabolism and function by a range of mechanisms involving numerous protein-protein and protein-RNA interactions. Such regulation has an important role in the context-specific functions of miRNAs.

Strengths and limitations of laboratory procedures for microRNA detection.
Koshiol J, Wang E, Zhao Y, Marincola F, Landi MT.
Infections and Immunepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Boulevard, Room 7070, Rockville, MD 20852-7248, USA
Cancer Epidemiol Biomarkers Prev. 2010 19(4): 907-911

BACKGROUND: MicroRNAs (miR) are endogenous, noncoding RNAs involved in many cellular processes and have been associated with the development and progression of cancer. There are many different ways to evaluate miRs.
METHODS: We described some of the most commonly used and promising miR detection methods.
RESULTS: Each miR detection method has benefits and limitations. Microarray profiling and quantitative real-time reverse-transcription PCR are the two most common methods to evaluate miR expression. However, the results from microarray and quantitative real-time reverse-transcription PCR do not always agree. High-throughput, high-resolution next-generation sequencing of small RNAs may offer the opportunity to quickly and accurately discover new miRs and confirm the presence of known miRs in the near future.
CONCLUSIONS: All of the current and new technologies have benefits and limitations to consider when designing miR studies. Results can vary across platforms, requiring careful and critical evaluation when interpreting findings.
IMPACT: Although miR detection and expression analyses are rapidly improving, there are still many technical challenges to overcome. The old molecular epidemiology tenet of rigorous biomarker validation and confirmation in independent studies remains essential.

Editorial  -  Studying microRNAs in the brain: technical lessons learned from the first ten years.
Hébert SS, Nelson PT.
Exp Neurol. 2012 235(2): 397-401

Pitfalls and recommendations  for microRNA expression  analysis using qPCR
Guidelines to microRNA qPCR with examples from the miRCURY LNA Universal RT microRNA PCR system
Exiqon Application Note

Introduction:  Over the last few years, qPCR has become the most widely used method for the study of microRNAs. It is fast, extremely sensitive and offers linear detection over several orders of magnitude. Exiqon’s miRCURY LNA™ Universal RT microRNA PCR system can profile microRNAs on panels in just three hours and offers a linear range of 7 orders of magnitude. The ingenious design, using LNA™ and two microRNA specific primers, allows individual microRNAs to be accurately quantified from as little as 1pg total RNA. This level of sensitivity enables microRNA profiling from difficult samples such as FFPE, LCM and body fluids including serum/plasma and urine. However, in order to get biologically relevant results, it is important to set up the qPCR experiment correctly.Even very small changes in microRNA expression levels, e.g. in comparing different disease stages, might be biologically significant. Yet, it will require a sufficient number of samples and correct normalization to reveal the differences with statistical significance. An insufficient number of replicates may obscure discovery of small but important differences. Poor normalization can lead to incorrect conclusions regarding the magnitude of regulation and even direction of fold change when studying differential expression. Proper study design and reliable normalization is therefore critical when analyzing differences in microRNA expression.In this document, we will go through the steps involved in setting up a qPCR study and explain how to perform the normalization and data analysis.

Potential pitfalls in microRNA profiling.
Chugh P, Dittmer DP.
Department of Microbiology, UNC-Chapel Hill, Chapel Hill, NC, USA.
Wiley Interdiscip Rev RNA. 2012 May 7

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally influence a wide range of cellular processes such as the host response to viral infection, innate immunity, cell cycle progression, migration, and apoptosis through the inhibition of target mRNA translation. Owing to the growing number of miRNAs and identification of their functional roles, miRNA profiling of many different sample types has become more expansive, especially with relevance to disease signatures. In this review, we address some of the advantages and potential pitfalls of the currently available methods for miRNA expression profiling. Some of the topics discussed include isomiRNAs, comparison of different profiling platforms, normalization strategies, and issues with regard to sample preparation and experimental analyses. WIREs RNA 2011 DOI: 10.1002/wrna.1120 For further resources related to this article, please visit the WIREs website.

Technolgy Feature -- MicroRNA profiling: separating signal from noise
Monya Baker
Various platforms for measuring microRNAs can provide different answers.

How Do MicroRNAs Regulate Gene Expression?
Richard J. Jackson & Nancy Standart
Sci STKE. 2007(367): re1
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road,
Cambridge CB2 1GA, UK

Several thousand human genes, amounting to aboutone-third of the whole genome, are potential targetsfor regulation by the several hundred microRNAs(miRNAs) encoded in the genome. The regulationoccurs posttranscriptionally and involves the ~21-nucleotide miRNAinteracting with a target site in themRNAthat generally has imperfect complementarityto the miRNA. The target sites are almost invariablyin the 3′-untranslated region of the messenger RNA(mRNA), often in multiple copies. Metazoan miRNAswere previously thought to down-regulate proteinexpression by inhibiting target mRNAtranslation atsome stage after the translation initiation step, with-out much effect on mRNAabundance. However,recent studies have questioned these suppositions.With some targets, an increase in the rate of mRNAdegradation by the normal decay pathway con-tributes to the decrease in protein expression.miRNAs can also inhibit translation initiation, specif-ically the function of the cap-binding initiation factor,eIF4E. Repressed target mRNAs as well as miRNAsthemselves accumulate in cytoplasmic foci knownas P-bodies, where many enzymes involved in mRNAdegradation are concentrated. However, P-bodiesmay also serve as repositories for the temporary andreversible storage of untranslated mRNA, and reduc-ing the expression (knockdown) of several distinctP-body protein components can alleviate miRNA-mediated repression of gene expression.
Normalization strategies for microRNA profiling experiments: a 'normal' way to a hidden layer of complexity?
Meyer SU, Pfaffl MW, Ulbrich SE
Biotechnol Lett. 2010 Aug 12
Physiology Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany

MicroRNA (miRNA) profiling is a first important step in elucidating miRNA functions. Real time quantitative PCR (RT-qPCR) and microarray hybridization approaches as well as ultra high throughput sequencing of miRNAs (small RNA-seq) are popular and widely used profiling methods. All of these profiling approaches face significant introduction of bias. Normalization, often an underestimated aspect of data processing, can minimize systematic technical or experimental variation and thus has significant impact on the detection of differentially expressed miRNAs. At present, there is no consensus normalization method for any of the three miRNA profiling approach. Several normalization techniques are currently in use, of which some are similar to mRNA profiling normalization methods, while others are specifically modified or developed for miRNA data. The characteristic nature of miRNA molecules, their composition and the resulting data distribution of profiling experiments challenges the selection of adequate normalization techniques. Based on miRNA profiling studies and comparative studies on normalization methods and their performances, this review provides a critical overview of commonly used and newly developed normalization methods for miRNA RT-qPCR, miRNA hybridization microarray, and small RNA-seq datasets. Emphasis is laid on the complexity, the importance and the potential for further optimization of normalization techniques for miRNA profiling datasets.

The microcosmos of cancer.
Lujambio A, Lowe SW.
Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA.
Nature. 2012 Feb 15;482(7385): 347-355

The discovery of microRNAs (miRNAs) almost two decades ago established a new paradigm of gene regulation. During the past ten years these tiny non-coding RNAs have been linked to virtually all known physiological and pathological processes, including cancer. In the same way as certain key protein-coding genes, miRNAs can be deregulated in cancer, in which they can function as a group to mark differentiation states or individually as bona fide oncogenes or tumour suppressors. Importantly, miRNA biology can be harnessed experimentally to investigate cancer phenotypes or used therapeutically as a target for drugs or as the drug itself.

miRNAs in human cancer.
Farazi TA, Spitzer JI, Morozov P, Tuschl T.
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, New York, NY 10065, USA.
J Pathol. 2011 Jan;223(2): 102-115

Mature microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotide (nt) length that control gene expression in many cellular processes. These molecules typically reduce the stability of mRNAs, including those of genes that mediate processes in tumorigenesis, such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis and invasion. miRNA targeting is mostly achieved through specific base-pairing interactions between the 5' end ('seed' region) of the miRNA and sites within coding and untranslated regions (UTRs) of mRNAs; target sites in the 3' UTR lead to more effective mRNA destabilization. Since miRNAs frequently target hundreds of mRNAs, miRNA regulatory pathways are complex. To provide a critical overview of miRNA dysregulation in cancer, we first discuss the methods currently available for studying the role of miRNAs in cancer and then review miRNA genomic organization, biogenesis and mechanism of target recognition, examining how these processes are altered in tumorigenesis. Given the critical role miRNAs play in tumorigenesis processes and their disease-specific expression, they hold potential as therapeutic targets and novel biomarkers.

MicroRNAs: genomics, biogenesis, mechanism, and function.
Bartel DP.
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
Cell. 2004 Jan 23;116(2): 281-297.

MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.

MicroRNA: past and present
Yang Wang, Heidi M. Stricker, Deming Gou, Lin Liu
Department of Physiological Sciences, Oklahoma State University, Stillwater, OK, 74078
Frontiers in Bioscience 12, 2316-2329, January 1, 2007

MicroRNAs (miRNAs) are ~22 nucleotide (nt) non-coding RNAs that participate in gene regulation.  MiRNAs confer their regulation at a post-transcriptional level, where they either cleave or repress translation of mRNAs.  Over 3000 identified mature miRNAs exist in species ranging from plants to humans, suggesting that they are ancient players in gene regulation.  A relatively small number of miRNAs have been experimentally tested for their function.  Of those tested, functions including cell differentiation, proliferation, apoptosis, anti-viral defense and cancer have been proposed.  Improved software programs are now able to predict the targets of miRNAs in a more efficient manner, thus facilitating the elucidation of miRNA function.  Furthermore, methods such as real-time PCR and microarray have been enhanced for studying miRNA expression.  Using these tools, scientists are able to discover novel functions for miRNAs.  It is possible that miRNAs will be revealed as having a role in virtually every aspect of gene regulation.  This review guides readers through the biogenesis of miRNAs, their mechanism of action on their target mRNAs, the functional outcomes of their action on mRNAs and the current techniques to investigate these processes.

Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?
Filipowicz W, Bhattacharyya SN, Sonenberg N.
Friedrich Miescher Institute for Biomedical Research, 4002 Basel, Switzerland
Nat Rev Genet. 2008 Feb;9(2): 102-114.

MicroRNAs constitute a large family of small, approximately 21-nucleotide-long, non-coding RNAs that have emerged as key post-transcriptional regulators of gene expression in metazoans and plants. In mammals, microRNAs are predicted to control the activity of approximately 30% of all protein-coding genes, and have been shown to participate in the regulation of almost every cellular process investigated so far. By base pairing to mRNAs, microRNAs mediate translational repression or mRNA degradation. This Review summarizes the current understanding of the mechanistic aspects of microRNA-induced repression of translation and discusses some of the controversies regarding different modes of microRNA function.

microRNAs in vertebrate physiology and human disease
Chang TC, Mendell JT.
The McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University
School of Medicine, Baltimore, Maryland, 21205, USA.
Annu Rev Genomics Hum Genet. 2007;8: 215-239.

Over the past five years, the importance of a diverse class of 18-24 nucleotide RNA molecules, known as microRNAs (miRNAs) has increasingly been recognized. These highly conserved RNAs regulate the stability and translational efficiency of complementary target messenger RNAs. The human genome is now predicted to encode nearly 1,000 miRNAs that likely regulate at least one third of all human transcripts. Despite rapid progress in miRNA discovery, the physiologic functions of only a small number have been definitively established. In this review, we discuss the principles of miRNA function that have emerged from the studies performed thus far in vertebrates. We also discuss known and potential roles for miRNAs in human disease states and discuss the influence of human genetic variation on miRNA-mediated regulation.

Illuminating the silence: understanding the structure and function of small RNAs
Tariq M. Rana

RNA interference (RNAi) is triggered by double-stranded RNA helices that have been introduced exogenously into cells as small interfering (si)RNAs or that have been produced endogenously from small non-coding RNAs known as microRNAs (miRNAs). RNAi has become a standard experimental tool and its therapeutic potential is being aggressively harnessed. Understanding the structure and function of small RNAs, such as siRNAs and miRNAs, that trigger RNAi has shed light on the RNAi machinery. In particular, it has highlighted the assembly and function of the RNA-induced silencing complex (RISC), and has provided guidelines to efficiently silence genes for biological research and therapeutic applications of RNAi.

Everything you wanted to know about small RNA but were afraid to ask
Boyd SD.
Department of Pathology, Stanford University School of Medicine, Stanford, CA
94305-2297, USA.
Lab Invest. 2008 Jun;88(6): 569-578

MicroRNAs are a class of recently discovered small RNA molecules that regulate other genes in the human genome. Studies in human cells and model organisms have begun to reveal the mechanisms of microRNA activity, and the wide range of normal physiological functions they influence. Their alteration in pathologic statesfrom cancer to cardiovascular disease is also increasingly clear. A review of current evidence for the role of these molecules in human health and disease will be helpful to pathologists and medical researchers as the fascinating story of these small regulators continues to unfold.

The regulation of genes andgenomes by small RNAs
Victor Ambros & Xuemei Chen
Development 134, 1635-1641(2007)

A recent Keystone Symposium on ‘MicroRNAs and siRNAs:Biological Functions and Mechanisms’ was organized by DavidBartel and Shiv Grewal (and was held in conjunction with ‘RNAifor Target Validation and as a Therapeutic’, organized byStephen Friend and John Maraganore). The ‘MicroRNAs andsiRNAs’ meeting brought together scientists working on diversebiological aspects of small regulatory RNAs, includingmicroRNAs, small interfering RNAs (siRNAs) and Piwi-interactingRNAs (piRNAs and rasiRNAs). Among the themes discussed werethe diversity of small regulatory RNAs and their developmentalfunctions, their biogenesis, the identification of their regulatorytargets, their mechanisms of action, and their roles in theelaboration of multicellular complexity.

MicroRNAs in Gene Regulation: When the Smallest Governs It All
Ouellet DL, Perron MP, Gobeil LA, Plante P, Provost P.
J Biomed Biotechnol. 2006;2006(4): 69616.

Encoded by the genome of most eukaryotes examined so far, microRNAs (miRNAs) are small ~21-nucleotide (nt) noncoding RNAs (ncRNAs) derived from a biosynthetic cascade involving sequential processing steps executed by the ribonucleases (RNases) III Drosha and Dicer. Following their recent identification, miRNAs have rapidly taken the center stage as key regulators of gene expression. In this review, we will summarize our current knowledge of the miRNA biosynthetic pathway and its protein components, as well as the processes it regulates via miRNAs, which are known to exert a variety of biological functions in eukaryotes. Although the relative importance of miRNAs remains to be fully appreciated, deregulated protein expression resulting from either dysfunctional miRNA biogenesis or abnormal miRNA-based gene regulation may represent a key etiologic factor in several, as yet unidentified, diseases. Hence is our need to better understand the complexity of the basic mechanisms underlying miRNA biogenesis and function.

Clustering and conservation patterns of human microRNAs
Altuvia Y, Landgraf P, Lithwick G, Elefant N, Pfeffer S, Aravin A, Brownstein MJ, Tuschl T, Margalit H.
Department of Molecular Genetics and Biotechnology, Faculty of Medicine, The
Hebrew University PO Box 12272, Jerusalem 91120, Israel.
Nucleic Acids Res. 2005 May 12;33(8): 2697-2706

MicroRNAs (miRNAs) are approximately 22 nt-long non-coding RNA molecules, believed to play important roles in gene regulation. We present a comprehensive analysis of the conservation and clustering patterns of known miRNAs in human. We show that human miRNA gene clustering is significantly higher than expected at random. A total of 37% of the known human miRNA genes analyzed in this study appear in clusters of two or more with pairwise chromosomal distances of at most 3000 nt. Comparison of the miRNA sequences with their homologs in four other organisms reveals a typical conservation pattern, persistent throughout the clusters. Furthermore, we show enrichment in the typical conservation patterns and other miRNA-like properties in the vicinity of known miRNA genes, compared with random genomic regions. This may imply that additional, yet unknown, miRNAs reside in these regions, consistent with the current recognition that there are overlooked miRNAs. Indeed, by comparing our predictions with cloning results and with identified miRNA genes in other mammals, we corroborate the predictions of 18 additional human miRNA genes in the vicinity of the previously known ones. Our study raises the proportion of clustered human miRNAs that are <3000 nt apart to 42%. This suggests that the clustering of miRNA genes is higher than currently acknowledged, alluding to its evolutionary and functional implications.

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