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) microDNAs
microRNAs -- overview
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.
Regulation of microRNA biogenesis
Ha M, Kim VN.
Nat Rev Mol Cell Biol. 2014 15(8): 509-524
MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins - Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.
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
microDNA -- A new piece of genetics puzzle
by DNA Decoding APRIL 15, 2012
In the beginning the big discovery was the existence of DNA and RNA. Eventually more refined experiments and better equipment revealed that RNA in particular came in many forms and functions, for example, micro RNA (miRNA) for DNA regulation or piwi-interacting RNA (piRNA) for transposon defense. So far there are 25-27 types of RNA. However, for DNA not so many types, in fact, basically two: chromosomal DNA, which is what most people think of as DNA, the DNA in the nucleus of every living cell. It comes with variants B (right handed helix twist) or A (right twist helix with 11 base pairs) and Z (left twist helix with 12 base pairs). Then there is mitochondrial DNA of the mitochondrion, the tiny enclosed organelle found in animal (eukaryote) cells. In short, the basic code storing function of DNA is in a relatively orderly format, whereas RNA the transcriber and regulator of DNA is very complex and geneticists continue to find more complications. Except that now there appears to be a new form of DNA, microDNA.
This new type of DNA is, for one thing, distinguished by existing outside the chromosome. Finding bits and pieces of DNA separated from the chromosome, in itself, isn’t too surprising. It’s a bit like finding flotsam along the shoreline; you expect some loose bits of material to be floating around in the cell. However, what scientists now call an extra-chromosomal circular DNA (eccDNA) may be something more significant.
One type of eccDNA, dubbed microDNA and recently discovered by scientists at the University of Virginia (USA) and the University of North Carolina (USA), is found in great numbers of relatively short strands (200-400 base pairs – the combinations of Guanine-Cytosine and Adenine-Thymine) in non-repeating sequences. Their finding has just been published in Science [08 March 2012, paywalled, Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues]. Where these ‘pieces’ of DNA come from has not been verified, but geneticists think it could be from cutting bits of chromosomal DNA (excision), replication of short DNA sequences, or reverse transcription of certain RNA. The research tends to show that microDNA mostly comes from deletions, which would indicate they are part of the repair and maintenance process for DNA.
The big question is what – if anything – are microDNA pieces for? Do they play an active role in the repair process, or are they the result (detritus) of that process? They do seem to be associated with gene variation between different types of cells. So far the researchers have found microDNA in human and mouse cells, but it may not be universal. At this point there are more questions than answers, although the pattern in genetic discovery tends to lead from the simple toward the complex. It is possible that microDNA and other eccDNAs have an important role in the genome – or not. It’s these kinds of questions that keep geneticists on their toes.Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues
Shibata Y, Kumar P, Layer R, Willcox S, Gagan JR, Griffith JD, Dutta A.
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA.
Science. 2012 Apr 6;336(6077): 82-86
We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200 to 400 base pairs long, are derived from unique nonrepetitive sequence, and are enriched in the 5'-untranslated regions of genes, exons, and CpG islands. Chromosomal loci that are enriched sources of microDNA in the adult brain are somatically mosaic for microdeletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a previously unknown DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.
A mechanism of gene amplification driven by small DNA fragments
Mukherjee K, Storici F.
School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.
PLoS Genet. 2012 8(12): e1003119
DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature.
microRNA -- Definition
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