microRNA Quality Control Study -- Hot papers:

Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study
Mestdagh P, Hartmann N, Baeriswyl L, Andreasen D, Bernard N, Chen C, Cheo D, D'Andrade P, DeMayo M, Dennis L, Derveaux S, Feng Y, Fulmer-Smentek S, Gerstmayer B, Gouffon J, Grimley C, Lader E, Lee KY, Luo S, Mouritzen P, Narayanan A, Patel S, Peiffer S, Rüberg S, Schroth G, Schuster D, Shaffer JM, Shelton EJ, Silveria S, Ulmanella U, Veeramachaneni V, Staedtler F, Peters T, Guettouche T1, Vandesompele J.
Nature Methods 11, 809–815 (2014)

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

RNA degradation compromises the reliability of microRNA expression profiling
David Ibberson, Vladimir Benes, Martina U Muckenthaler and Mirco Castoldi
1 Genomics Core Facility, EMBL, Meyerhofstraße 1 D-69117 Heidelberg, Germany; 2 Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 156, D-69120, Heidelberg, Germany; 3 Molecular Medicine Partnership Unit, Im Neuenheimer Feld 156, D-69120, Heidelberg, Germany
BMC Biotechnology 2009, 9:102doi:10.1186/1472-6750-9-102

MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles. Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP) or microRNA-specific real-time quantitative PCR (miQPCR). Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays. MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven.

mRNA and microRNA quality control for RT-qPCR analysis
Becker C, Hammerle-Fickinger A, Riedmaier I, Pfaffl MW.
Physiology-Weihenstephan, Technical University Munich, Freising, Germany.
Methods. 2010 Apr;50(4):237-43. Epub 2010 Jan 15.

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency. RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity.

A comparison of miRNA isolation and RT-qPCR technologies and their effects on quantification accuracy and repeatability.
Redshaw N, Wilkes T, Whale A, Cowen S, Huggett J, Foy CA.
LGC Limited, Queens Road, Teddington, Middlesex, UK.
Biotechniques. 2013 54(3): 155-164

MicroRNAs (miRNAs) are short (~22 nucleotides), non-coding RNA molecules that post-transcriptionally regulate gene expression. As the miRNA field is still in its relative infancy, there is currently a lack of consensus regarding optimal methodologies for miRNA quantification, data analysis and data standardization. To investigate miRNA measurement we selected a panel of both synthetic miRNA spikes and endogenous miRNAs to evaluate assay performance, copy number estimation, and relative quantification. We compared two different miRNA quantification methodologies and also assessed the impact of short RNA enrichment on the miRNA measurement. We found that both short RNA enrichment and quantification strategy used had a significant impact on miRNA measurement. Our findings illustrate that miRNA quantification can be influenced by the choice of methodology and this must be considered when interpreting miRNA analyses. Furthermore, we show that synthetic miRNA spikes can be used as effective experimental controls for the short RNA enrichment procedure.

Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses.
Hammerle-Fickinger A, Riedmaier I, Becker C, Meyer HH, Pfaffl MW, Ulbrich SE.
Biotechnol Lett. 2010 32(1): 35-44 Supplement - Biotechnol Lett. 2010 32(1): 35-44
Physiology Weihenstephan, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354, Freising, Germany.

The benefit and precision of blood diagnosis by quantitative real-time PCR (qPCR) is limited by sampling procedures and RNA extraction methods. We have compared five different RNA extraction protocols from bovine blood regarding RNA and miRNA yield, quality, and most reproducible data in the qRT-PCR with the lowest point of quantification. Convincing results in terms of highest quantity, quality, and best performance for mRNA qPCR were obtained by leukocyte extraction following blood lysis as well as extraction of PAXgene stabilized blood. The best microRNA qPCR results were obtained for samples extracted by the leukocyte extraction method.

RNA quality control in miRNA expression analysis
Christiane Becker, Martina Reiter, Michael W. Pfaffl
Agilent Technologies Application Note 5990-5557EN

It is generally known that total RNA quality has a distinct influence on the validity and reliability of quantitative PCR results. In addition, the recently published MIQE guidelines focus on the pre-PCR steps and state the importance of RNA quality assessment. Various studies showed the impairing effect of ongoing RNA degradation on mRNA expression results. Therefore, the verification of RNA integrity prior to downstream applications like RT-qPCR and mircroarrays is indispensable. A fast and reliable assessment of RNA integrity can be done with the Eukaryote Total RNA Nano Assay of the Agilent 2100 Bioanalyzer. The importance of RNA quality should also be considered in new applications such as the investigation of miRNA expression profiles. With the Agilent Small RNA Assay, Agilent is offering one of the few possibilities for selectively estimating miRNA before expression analysis. However, by now little is known about factors affecting miRNA analysis. Herein, the important impact of total RNA quality on quantification of mRNA and miRNA should be considered.


	MicroRNAs (miRNAs) - Nature Supplement December 2009 http://www.nature.com/nature/supplements/collections/micrornas/ MicroRNAs are small, non–coding RNAs found in plants and animals. They regulate gene expression by binding to complementary sequences within target mRNAs. The mammalian genome encodes hundreds of miRNAs that collectively affect the expression of about one–third of all genes. This collection showcases the latest papers from Nature that explore the biogenesis, biological effects in both normal and diseased cells, and therapeutic potential of miRNAs.

	Nature Genetics 38, S1 (2006) The microRevolution microRNAs (miRNAs) were initially considered a biological sideshow, the oddly interesting regulators of developmental timing genes in Caenorhabditis elegans. But in the past few years, studies have shown that miRNAs are a considerable part of the transcriptional output of the genomes of plants and animals, that they regulate a large part of their transcriptomes and that they serve important regulatory functions in widespread biological activities. Accordingly, miRNAs are now recognized as an additional layer of post-transcriptional control that must be accounted for if we are to understand the complexity of gene expression and the regulatory potential of the genome. Owing to this impressive progress in understanding the genomics and functions of miRNAs, we think this is an ideal time to examine the available evidence to see where this rapidly growing field is going.
In this Supplement, we have focused on approaches to detect the presence of miRNAs and their impact on genomes, and we explore the roles they play in regulating biological functions. The Supplement consists of five exploratory Perspectives and a comprehensive Review; the pieces generally follow a progressive logic from discovery to target prediction to function to systems perspective and finally to organismal perspective. Plant and animal genomes have been shaped by miRNAs, as seen by the substantial number of conserved miRNAs that have accumulated through selection and the presence of miRNA target sites in genes of diverse functions. However, the true number of miRNAs and targets remains difficult to estimate. The detection of miRNAs is addressed in a Perspective from Eugene Berezikov, Edwin Cuppen and Ronald Plasterk (p S2), who discuss methods, both experimental and bioinformatic, for discovering new miRNAs. These authors wrangle with the question of how we define a 'true' miRNA and the implications this definition will have for future studies. Approaches to the prediction of targets of miRNAs are addressed by Nikolaus Rajewsky (p S8), who considers the case for combinatorial control of target expression by multiple miRNAs acting synergistically. Some of the fundamental goals of investigations into genome function are to understand how the genome gives rise to different cell types, how it contributes to basic and specialized functions in those cells and how it contributes to the ways cells interact with the environment. The roles of miRNAs in each of those functions are touched on in three Perspectives. Jan Krützfeldt, Matthew Poy and Markus Stoffel (p S14) discuss approaches and technological advances useful to the investigator studying miRNA function. Eran Hornstein and Noam Shomron (p S20) take a systems approach to conceptualize a network of interacting miRNAs and targets and propose that miRNAs act to canalize developmental gene expression programs. And Bryan Cullen (p S25) discusses recent evidence for pathogenic roles of virally encoded miRNAs and proposes that cellular miRNAs influence the cell-type specificity of invading viruses. In the last piece, Allison Mallory and Hervé Vaucheret (p S31) offer a view of the diverse biological roles of miRNAs from an organismal perspective in their Review of miRNAs and other endogenous regulatory RNAs in plants. This piece highlights the contributions of regulatory RNAs to developmental programs and stress responses. Our hope is that you find strategic advice and insight in this Supplement. We invite you to access its contents online at http://www.nature.com/ng/supplements/, where it will be freely available for 3 months. In addition to the pieces featured here, online we provide links to related articles on miRNAs published by the Nature Publishing Group and an animation entitled 'Lifecycle of an miRNA' supplied by Rosetta Genomics. We are grateful to our authors for their insightful contributions, as well as to our referees for their valuable comments during the review process. In addition, we gratefully acknowledge the support of our principal sponsors, Rosetta Genomics and Alnylam Pharmaceuticals, and our supporting sponsor, Santaris Pharma, for their help in producing this Supplement and making it freely available online.

	www.nature.com/reviews/focus/microrna/index.html Nature Reviews presents a Collection on microRNAs, which includes Reviews from Nature Reviews Genetics, Nature Reviews Cancer and Nature Reviews Molecular Cell Biology. The articles have been specially selected to provide an introduction to diverse aspects of microRNA biology, including their biogenesis, function in normal development and cancer, and evolutionary implications of their impact on gene regulation.
The evolution of gene regulation by transcription factors and microRNAs Kevin Chen & Nikolaus Rajewsky; Nature Reviews Genetics 8, 93-103 (2007) MicroRNAs: small RNAs with a big role in gene regulation Lin He & Gregory J Hannon; Nature Reviews Genetics 5, 631 (2004) MicroRNA signatures in human cancers George A. Calin & Carlo M. Croce; Nature Reviews Cancer 6, 857-866 (2006) Oncomirs — microRNAs with a role in cancer Aurora Esquela-Kerscher & Frank J. Slack; Nature Reviews Cancer 6, 259-269 (2006) MicroRNA biogenesis: coordinated cropping and dicing V. Narry Kim; Nature Reviews Molecular Cell Biology 6, 376-385 (2005)

miRNA Research Education Research Center
by Agilent Technologies

Introduction

In 1993, R.C. Lee of Harvard University first described miRNA-mediated silencing in C. elegans, and since, these molecules have been more clearly defined as single-stranded RNA molecules, 19-25 nucleotides in length, that are generated from endogenous hairpin transcripts. MicroRNAs (miRNAs) serve as guides in post-transcriptional gene silencing by complimentary base pairing with target mRNAs, resulting in mRNA cleavage or translational repression. As a result, miRNAs enable regulation of complex biological pathways such as those associated with developmental processes, haematopoietic cell differentiation, apoptosis, and cell proliferation. Interestingly, it now appears that miRNAs may actually form complex regulatory networks with target mRNAs, as a single miRNA may be responsible for the regulation of several different targets, or conversly, several miRNAs may cooperatively regulate a single mRNA target. To date, there have been approximately 4300 precursor miRNAs found in virtually all species—animals, plants, and viruses—of which ~475 are human miRNAs. Research suggests that as many as one-third of all human genes may be miRNA regulated, many of which are involved in cancer and other disease regulation.

Due to their involvement in gene regulation, miRNAs have received significant attention with respect to their role in cancer, disease, and stem cell differentiation. Traditional characterization of miRNA follows small RNA identification by cDNA cloning. Expression of miRNAs is typically confirmed by hybridization to a size-fractionated RNA sample, usually achieved by Nothern blot analysis. Alternative methods for miRNA detection and confirmation include reverse transcription PCR (RT-PCR), primer extension analysis, RNase protection assays, and microarray analysis. Typically, even when miRNAs are identified using these alternative methods, Northern blot analysis follows as it enables the confirmation of both the hairpin precursor (~70 nt) and mature miRNA (~22 nt) forms. Global gene expression profiling of miRNAs using microarrays provides high-throughput information on miRNA involvement in disease progression and developmental changes, while offering an alternative to some of the time- and labor-intensive techniques previously described.

Cancer genomics: Small RNAs with big impacts

Paul S. Meltzer
Nature 435, 745-746 (9 June 2005) | doi: 10.1038/435745a

Although they are tiny, microRNAs can have large-scale effects because they regulate a variety of genes. These minuscule molecules are now definitively linked to the development of cancer. During the past few years, molecular biologists have been stunned by the discovery of hundreds of genes that encode small RNA molecules. These microRNAs (miRNAs -21 to 25 nucleotides in length)- are negative regulators of gene expression. The mechanisms by which they work are similar in plants and animals, implying that they are involved in fundamental cellular processes. As cancer is essentially a consequence of disordered genome function, one might expect these regulatory molecules to be involved in the development of this disease. Indeed, there are hints that the levels of some miRNAs are altered in cancer; there is also evidence that an miRNA regulates the cancer-promoting ras gene. Three studies in this issue change the landscape of cancer genetics by establishing the specific miRNAs expressed in most common cancers, and investigating the effects of miRNAs on cancer development and cancer genes..............

The precursor of an miRNA (pri-miRNA) is transcribed in the nucleus. It forms a stem−loop structure that is processed to form another precursor (pre-miRNA) before being exported to the cytoplasm. Further processing by the Dicer protein creates the mature miRNA, one strand of which is incorporated into the RNA-induced silencing complex (RISC). Base pairing between the miRNA and its target directs RISC to either destroy the mRNA or impede its translation into protein. The initial stem−loop configuration of the primary transcript provides structural clues that have been used to guide searches of genomic sequence for candidate miRNA genes.

MicroRNA in cancer

http://www.nature.com/nature/journal/v435/n7043/edsumm/e050609-16.html

MicroRNAs are regulatory, non-coding RNAs about 22 nucleotides in length: over 200 have been identified in humans, and their functions are beginning to be pinned down. It has been suggested that like other regulatory molecules they might be involved in tumour formation, and three papers in this issue confirm that this is the case. One cluster of microRNAs, known as mir-17−92, is shown to be a potential oncogene by its action in an in vivo model of human B-cell lymphoma. A cluster of microRNAs on human chromosome 13 has been found to be regulated by c-Myc, an important transcription factor that is overexpressed in many human cancers. And analysis of microRNA expression in over 300 individuals shows that microRNA profiles could be of value in cancer diagnosis. There is a global downregulation of microRNA in tumours, and the microRNA profile also reflects the origin and differentiation state of the tumours.

Letter: A microRNA polycistron as a potential human oncogene

Lin He, J. Michael Thomson, Michael T. Hemann, Eva Hernando-Monge, David Mu, Summer Goodson, Scott Powers, Carlos Cordon-Cardo, Scott W. Lowe, Gregory J. Hannon and Scott M. Hammond

Letter: MicroRNA expression profiles classify human cancers

Jun Lu, Gad Getz, Eric A. Miska, Ezequiel Alvarez-Saavedra, Justin Lamb, David Peck, Alejandro Sweet-Cordero, Benjamin L. Ebert, Raymond H. Mak, Adolfo A. Ferrando, James R. Downing, Tyler Jacks, H. Robert Horvitz and Todd R. Golub

Letter: c-Myc-regulated microRNAs modulate E2F1 expression

Kathryn A. O'Donnell, Erik A. Wentzel, Karen I. Zeller, Chi V. Dang and Joshua T. Mendell

Sizing up miRNAs as cancer genes

Carlos Caldas & James D Brenton

The authors are in the Cancer Genomics Program, Department of Oncology, University of Cambridge and Cambridge University Hospitals National Health Service Foundation Trust, Cambridge CB2 2XZ, UK.

Findings over the last year or so have built the case that microRNAs might contribute to cancer. Three studies now definitively show this to be the case and also suggest that these small RNAs could be used to categorize tumors.

Review: microRNAs as oncogenes and tumor suppressors.
Baohong Zhang, Xiaoping Pan, George P. Cobb, Todd A. Anderson
Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX 79409-1163, USA

microRNAs (miRNAs) are a new class of non-protein-coding, endogenous, small RNAs. They are important regulatory molecules in animals and plants. miRNA regulates gene expression by translational repression, mRNA cleavage, and mRNA decay initiated by miRNA-guided rapid deadenylation. Recent studies show that some miRNAs regulate cell proliferation and apoptosis processes that are important in cancer formation. By using multiple molecular techniques, which include Northern blot analysis, real-time PCR, miRNA microarray, up- or down-expression of specific miRNAs, it was found that several miRNAs were directly involved in human cancers, including lung, breast, brain, liver, colon cancer, and leukemia. In addition, some miRNAs may function as oncogenes or tumor suppressors. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs may play a more important role in the pathogenesis of a limited range of human cancers than previously thought. Overexpressed miRNAs in cancers, such as mir-17–92, may function as oncogenes and promote cancer development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation or apoptosis. Underexpressed miRNAs in cancers, such as let-7, function as tumor suppressor genes and may inhibit cancers by regulating oncogenes and/or genes that control cell differentiation or apoptosis. miRNA expression profiles may become useful biomarkers for cancer diagnostics. In addition, miRNA therapy could be a powerful tool for cancer prevention and therapeutics.

miRU: an automated plant miRNA target prediction server.

http://bioinfo3.noble.org/miRU.htm

Zhang Y.
Nucleic Acids Res. 2005 33 (Web Server issue)

Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA

MicroRNAs (miRNAs) play important roles in gene expression regulation in animals and plants. Since plant miRNAs recognize their target mRNAs by near-perfect base pairing, computational sequence similarity search can be used to identify potential targets. A web-based integrated computing system, miRU, has been developed for plant miRNA target gene prediction in any plant, if a large number of sequences are available. Given a mature miRNA sequence from a plant species, the system thoroughly searches for potential complementary target sites with mismatches tolerable in miRNA-target recognition. True or false positives are estimated based on the number and type of mismatches in the target site, and on the evolutionary conservation of target complementarity in another genome which can be selected according to miRNA conservation. The output for predicted targets, ordered by mismatch scores, includes complementary sequences with mismatches highlighted in colors, original gene sequences and associated functional annotations.
The miRU web server is available at http://bioinfo3.noble.org/miRU.htm

The RNAi Consortium shRNA Library

http://www.broad.mit.edu/genome_bio/trc/rnai.html

The RNAi Consortium, or TRC, is a public-private effort based at the Broad whose mission is to create a shRNA library as well to validate tools and methods that will enable the scientific community to use RNAi to determine the function of human and mouse genes. The reagents are composed of short hairpin sequences carried in lentiviral vectors arrayed in 96-well plates.

The RNAi Consortium - * Details on the TRC shRNA Library

Since RNA interference (RNAi) was discovered to work in mammalian cells, the genetic manipulation technique has been hailed as a revolutionary new approach to basic biological research and drug development and discovery. RNAi is expected to provide critical insights into the mechanisms underlying human disease and accelerating development of treatments for cancer, AIDS and a host of other disorders.
A public-private consortium based at the Broad will develop and validate tools and methods that will enable the worldwide scientific community to use RNAi to unveil the function of most human and mouse genes. The goal of the RNAi Consortium (abbreviated TRC) is to use the recently discovered RNAi mechanism to create widely applicable research reagents consisting of specific inhibitors against human and mouse genes. The reagents are composed of short hairpin sequences carried in lentiviral vectors. They can be used in a wide range of cellular and animal studies to discover the key genes underlying normal physiology and disease.
In a three-year, $18 million initiative, the TRC will create a library of materials to conduct RNAi experiments on 15,000 human genes and 15,000 mouse genes. A total of 150,000 custom-designed plasmids that express short and unique pieces of RNA (known as short hairpin RNAs or shRNAs) that target specific genes will be created and validated. This fundamental resource will be made available to scientists worldwide through commercial and academic distributors.
In addition to the Broad, TRC partners include Harvard Medical School (HMS), the Massachusetts Institute of Technology (MIT), Dana-Farber Cancer Institute (DFCI), the Whitehead Institute for Biomedical Research (WIBR), Novartis, Eli Lilly, Bristol-Myers Squibb, Sigma-Aldrich and research institute Academia Sinica in Taiwan.
The Principal Investigators of TRC are Dr. Nir Hacohen (Broad Associate Member; Massachusetts General Hospital, HMS), Dr. William Hahn (Broad Associate Member; DFCI, HMS), Dr. Eric Lander (Broad Institute), Dr. David Root (TRC Project Manager, Broad Institute), Dr. David Sabatini (Broad Associate Member; WIBR, MIT), Dr. Sheila Stewart (Washington University, formerly at WIBR), and Dr. Brent Stockwell (Columbia University, formerly at WIBR).

M. Zuker - Research
Algorithms & Thermodynamics for Nucleic Acid Folding, Hybridization & Melting Profile Prediction

http://frontend.bioinfo.rpi.edu/zukerm/cgi-bin/rna-index.cgi

http://www.bioinfo.rpi.edu/applications/mfold

Mfold web server for nucleic acid folding and hybridization prediction.
M. Zuker Nucleic Acids Res. 31 (13), 3406-15, (2003)

The abbreviated name, ‘mfold web server’, describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and ‘energy dot plots’, are available for the folding of single sequences. A variety of ‘bulk’ servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold This URL will be referred to as ‘MFOLDROOT’.

Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure

David H. Mathews1, Jeffrey Sabina1, Michael Zuker2 and Douglas H. Turner1

1 Department of Chemistry University of Rochester, Rochester, NY 14627-0216, USA
2 Institute for Biomedical Computing, Washington University, St. Louis MO 63110, USA

An improved dynamic programming algorithm is reported for RNA secondary structure prediction by free energy minimization. Thermodynamic parameters for the stabilities of secondary structure motifs are revised to include expanded sequence dependence as revealed by recent experiments. Additional algorithmic improvements include reduced search time and storage for multibranch loop free energies and improved imposition of folding constraints. An extended database of 151,503 nt in 955 structures? determined by comparative sequence analysis was assembled to allow optimization of parameters not based on experiments and to test the accuracy of the algorithm. On average, the predicted lowest free energy structure contains 73 % of known base-pairs when domains of fewer than 700 nt are folded; this compares with 64 % accuracy for previous versions of the algorithm and parameters. For a given sequence, a set of 750 generated structures contains one structure that, on average, has 86 % of known base-pairs. Experimental constraints, derived from enzymatic and flavin mononucleotide cleavage, improve the accuracy of structure predictions.

miRBase - the home of microRNA data

miRBase http://microrna.sanger.ac.uk/ is the new home of microRNA data on the web, providing data previously accessible from the miRNA Registry. Old miRNA Registry addresses should redirect you to this page.

The miRBase Sequence Database is a searchable database of published miRNA sequences and annotation. The data were previously provided by the miRNA Registry.
The miRBase Registry continues to provide gene hunters with unique names for novel miRNA genes prior to publication of results.
The miRBase Targets database is a new resource of predicted miRNA targets in animals.

Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching using BLAST and SSEARCH, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download.

Please note that the predicted stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), but include the pre-miRNA and some flanking sequence from the presumed primary transcript.

Please use the tabs along the top of this page to access the different areas of the site, or you can click here to jump to the help pages. A summary of the data available in the current release is provided here.

To receive email notification of data updates and feature changes please subscribe to the microRNA mailing list. Any other queries about the website or naming service should be directed at microRNA@sanger.ac.uk.

References: If you make use of the data presented here, please cite the following articles in addition to the primary data sources

miRBase: microRNA sequences, targets and gene nomenclature

Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ.
NAR, 2006, 34, Database Issue, D140-D144

The miRBase database aims to provide integrated interfaces to comprehensive microRNA sequence data, annotation and predicted gene targets. miRBase takes over functionality from the microRNA Registry and fulfils three main roles: the miRBase Registry acts as an independent arbiter of microRNA gene nomenclature, assigning names prior to publication of novel miRNA sequences. miRBase Sequences is the primary online repository for miRNA sequence data and annotation. miRBase Targets is a comprehensive new database of predicted miRNA target genes.

The microRNA Registry

Griffiths-Jones S.
NAR, 2004, 32, Database Issue, D109-D111

The miRNA Registry provides a service for the assignment of miRNA gene names prior to publication. A comprehensive and searchable database of published miRNA sequences is accessible via a web interface (http://www.sanger.ac.uk/Software/Rfam/mirna/), and all sequence and annotation data are freely available for download. Release 2.0 of the database contains 506 miRNA entries from six organisms.

The following publication provides guidelines on miRNA annotation:

A uniform system for microRNA annotation

Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T.
RNA, 2003, 9(3), 277-279

MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. Guidelines are presented for the identification and annotation of new miRNAsfrom diverse organisms, particularly so that miRNAs can be reliably distinguished from other RNAs such as small interfering RNAs. We describe specific criteria for the experimental verification of miRNAs, and conventions for naming miRNAs and miRNA genes. Finally, an online clearinghouse for miRNA gene name assignments is provided by the Rfam database of RNA families.