microRNA (miRNA) & quantitative real-time RT-PCR (1)
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 REVIEWS (6) UPDATED
microRNA normalisation (7) UPDATED
RNA interference (RNAi) small inhibiting RNA (siRNA) small activating RNA (saRNA)
The art of microRNA research
van Rooij E.
miRagen Therapeutics Inc., 6200 Lookout Road, Boulder, CO 80301, USA
Circ Res. 2011 108(2): 219-234.
Originally identified as moderate biological modifiers, microRNAs have recently emerged as powerful regulators of diverse cellular processes with especially important roles in disease and tissue remodeling. The rapid pace of studies on microRNA regulation and function necessitates the development of suitable techniques for measuring and modulating microRNAs in different model systems. This review summarizes experimental strategies for microRNA research and highlights the strengths and weaknesses of different approaches. The development of more specific and sensitive assays will further illuminate the biology behind microRNAs and will advance opportunities to safely pursue them as therapeutic modalities.
This animation describes Exiqon's LNA™ technology, and why it is superior to DNA in the study of microRNAs, which are challenging for many reasons => show animation
Their short length and the high sequence similarity between closely related microRNAs makes it hard to detect them with sufficient specificity and sensitivity. => Exiqon ProbeLibrary real-time PCR Assay System
Small non-coding RNAs in animal development
Giovanni Stefani & Frank J. Slack
Nature Reviews Molecular Cell Biology 9, 219-230 (2008)
The modulation of gene expression by small non-coding RNAs is a recently discovered level of gene regulation in animals and plants. In particular, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) have been implicated in various aspects of animal development, such as neuronal, muscle and germline development. During the past year, an improved understanding of the biological functions of small non-coding RNAs has been fostered by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators of animal development and are potential human disease loci.
In genetics, a miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotides long, and is thought to regulate the expression of other genes. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. The DNA sequence that codes for an miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA).
MicroRNAs are transcribed as long RNA precursors (pri-miRNAs) that contain a stem-loop structure of about 80 bases. Pri-miRNAs are processed in the nucleus by the RNase III enzyme Drosha and DGCR8/Pasha, which excises the stem-loop to form the pre-miRNA. Pre-miRNAs are exported from the nucleus by Exportin-5, a carrier protein. In the cytoplasm another RNase III enzyme, Dicer, cuts the pre-miRNA to generate the mature microRNA as part of a short RNA duplex. The RNA is subsequently unwound by a helicase activity and incorporated into a RNA induced silencing complex (RISC).
Most microRNAs in animals are thought to function through the inhibition of effective mRNA translation of target genes through imperfect base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs. However, the underlying mechanism is poorly understood. MicroRNA targets are largely unknown, but estimates range from one to hundreds of target genes for a given microRNA, based on target predictions using a variety of bioinformatics. In addition, at least one microRNA, miR-196, can cleave a target mRNA, HOXB8, like a siRNA. This mechanism is the preferred one for plant microRNAs. MicroRNAs may also play a role in AU-rich element-mediated mRNA degradation. Finally, microRNAs may also play roles in transcriptional gene silencing (TGS), which has been observed in plants.
In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA. The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5, a carrier protein. The Dicer enzyme cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.
The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA. This is thought to be the primary mode of action of plant miRNAs. In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi), though in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded. miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs. miRNAs function in association with a complement of proteins collectively termed the miRNP.
This effect was first described for the worm Caenorhabditis elegans in 1993 by R. C. Lee of Harvard University. As of 2002, miRNAs have been confirmed in various plants and animals, including C. elegans, human and the plant Arabidopsis thaliana. Genes have been found in bacteria that are similar in the sense that they control mRNA abundance or translation by binding an mRNA by base pairing, however they are not generally considered to be miRNAs because the Dicer enzyme is not involved.
The term miRNA was first introduced in a set of three articles in Science (26 October 2001)
In plants, similar RNA species termed short-interfering RNAs siRNAs are used to prevent the transcription of viral RNA. While this siRNA is double-stranded, the mechanism seems to be closely related to that of miRNA, especially taking the hairpin structures into account. siRNAs are also used to regulate cellular genes, as miRNAs do.
activity of an miRNA can be experimentally blocked using a locked
nucleic acid oligo, a Morpholino oligo or a 2'-O-methyl RNA oligo. Most
efficient methods for miRNA detection are based on oligonucleotides
modified with locked nucleic acids.
Non-coding RNA genes include transfer RNA (tRNA) and ribosomal RNA (rRNA), small RNAs such as snoRNAs, microRNAs, siRNAs and piRNAs and lastly long ncRNAs that include examples such as Xist, Evf, Air, CTN and PINK. The number of ncRNAs encoded within the genome is unknown, however recent transcriptomic and microarray studies suggest the existence of over 30,000 long ncRNAs and at least as many small regulatory RNAs within the mouse genome alone. Since most of the newly identified ncRNAs have not been validated for their function, it is possible that the majority of them are meaningless (e.g. non-functional or truncated transcript).
One of the major findings of the 2007 ENCODE Pilot Project was that "nearly the entire genome may be represented in primary transcripts that extensively overlap and include many non-protein-coding regions."
Types of non-coding RNAs
News and Views Q&A (by Helge Großhans and Witold Filipowicz)
Molecular biology -- The expanding world of small RNAs
Molecular cell biology has long been dominated by a protein-centric view. But the emergence of small, non-coding RNAs challenges this perception. These plentiful RNAs regulate gene expression at different levels, and have essential roles in health and disease.
POSTER -- Regulation of microRNA biogenesis, function and degradation
Jacek Krol, Inga Loedige and Witold Filipowicz
MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that are ~21-nucleotides in length and control many developmental and cellular processes in eukaryotes. The implication of miRNAs in many disease processes also makes them important potential targets for therapy. Research during the last decade has identified many of the components that participate in miRNA biogenesis and has established basic principles of miRNA function1. More recently, it has become apparent that miRNA regulators themselves are subject to sophisticated control. Many studies over the last few years have reported the regulation of miRNA biogenesis, function and degradation by a range of mechanisms involving numerous proteinprotein and protein-RNA interactions2. Such regulation has an important role in the context-specific functions of miRNAs and an understanding of this control is needed to gain a full picture of the roles of miRNAs in development, physiology and disease.
MicroRNA Target Prediction
miRanda — miRNA target prediction for human, drosophila and zebrafish genomes
miRBase — a comprehensive repository for miRNAs and their predicted targets
miRDB — an online database for miRNA target prediction and functional annotations in animals
miRNAMap — a genomic maps of microRNA genes and their target genes in mammalian genomes
miR2Disease — a database providing comprehensive resource of miRNA deregulation in various human diseases
TarBase — a comprehensive database of experimentally supported animal microRNA targets
PicTar — microRNA targets for vertebrates, fly and nematodes
TargetScan — a search for the presence of conserved sites that match the seed of each miRNA
Target Gene Prediction at EMBL — miRNA-Target predictions for Drosophila miRNAs
Databases for microRNA Expression
microRNA.org — predicted microRNA targets & target downregulation scores. Experimentally observed expression patterns
HMDD — Human MicroRNA Disease Database (HMDD) is a database that contains the experimentally supported miRNA-disease association data, which are manually curated from publications. The dysfunction evidence or miRNAs
and literature PubMed ID are also given
TransmiR — a web query-driven database integrating the experimentally supported transcription factor and miRNA regulatory relations
RNA Secondary Structure Prediction
DIANA MicroTest — a prediction of miRNA-mRNA interaction
mfold — tools for predicting the secondary structure of RNA and DNA, mainly by using thermodynamic methods
microInspector — a web tool for detection of miRNA binding sites in an RNA sequence
miRNA Bioinfor — miRNA End Energy calculator which takes miRNA duplex to calculate free energy for 5 base pairs at one end plus a dangling nucleotide
miRRim — a method for detecting miRNA foldbacks based on hidden Markov model (HMM)
MXSCARNA — a multiple alignment tool for RNA sequences using progressive alignment based on pairwise structural alignment algorithm of SCARNA. Good for large scale analyses.
RNAhybrid — a tool for finding the minimum free energy hybridisation of a long and a short RNA
MicroRNA Homologous Prediction
miRNAminer — a web-based tool used for homologous miRNA gene search in several species
miRviewer — a global view of homologous miRNA genes in many species
RISCbinder — prediction of guide strand of microRNAs
Mireval — Sequence evaluation of microRNA properties
MicroRNA Deep Sequencing
miRanalyzer — A microRNA detection and analysis tool for next-generation sequencing experiments
miRNAkey — A software pipeline for the analysis of microRNA Deep Sequencing data
miRDeep — Discovering known and novel miRNAs from deep sequencing data
Non-coding RNA database:
conjunction with the RIKEN and Karolinska Institutes, the IMB has
developed a comprehensive mammalian noncoding RNA database (RNAdb)
which contains over 800 unique experimentally studied noncoding RNAs,
including many associated with diseases and/or developmental processes.
The database includes microRNAs and snoRNAs, but not infrastructural
RNAs such as rRNAs and tRNAs which are catalogued elsewhere. The
database also includes over 1200 putative antisense ncRNAs and almost
20,000 putative noncoding RNAs identified in high quality murine and
human cDNA libraries, with more to be added in the near future. Many of
these RNAs are large, and many are spliced, some alternatively. For
ncRNAs listed in RNAdb, sequence data as well as other information
including Genbank accessions, references, chromosomal location,
transcript length, splicing status, conservation notes, function,
disease associations, antisense relationships, imprinting status, and
tissue expression patterns are provided wherever possible. The database
is searchable by many criteria, and will we hope be useful as a
foundation for the emerging field of RNomics and the characterization
of the roles of ncRNAs in mammalian gene expression and regulation.
In conjunction with the RIKEN and Karolinska Institutes, the IMB has developed a comprehensive mammalian noncoding RNA database (RNAdb) which contains over 800 unique experimentally studied noncoding RNAs, including many associated with diseases and/or developmental processes. The database includes microRNAs and snoRNAs, but not infrastructural RNAs such as rRNAs and tRNAs which are catalogued elsewhere. The database also includes over 1200 putative antisense ncRNAs and almost 20,000 putative noncoding RNAs identified in high quality murine and human cDNA libraries, with more to be added in the near future. Many of these RNAs are large, and many are spliced, some alternatively.
For ncRNAs listed in RNAdb, sequence data as well as other information including Genbank accessions, references, chromosomal location, transcript length, splicing status, conservation notes, function, disease associations, antisense relationships, imprinting status, and tissue expression patterns are provided wherever possible. The database is searchable by many criteria, and will we hope be useful as a foundation for the emerging field of RNomics and the characterization of the roles of ncRNAs in mammalian gene expression and regulation.
If you find this website useful and would like to cite RNAdb, please use the following reference:
Stephen, S., Engstrom, P.G., Tajul-Arifin, K., Chen, W., Wahlestedt,
C., Lenhard, B., Hayashizaki, Y., Mattick, J.S. (2005)
RNAdb - a comprehensive mammalian noncoding RNA database.
Res. 33 (Database Issue): D125-130