 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 REVIEWS & microRNA normalisation (6) Quantitative real-time RT-PCR applications for microRNA quantification:
Editorial
- The microRevolution
NATURE GENETICS SUPPLEMENT | VOLUME 38 | JUNE 2006 P E R S P E C T I V E S : MOLECULAR BIOLOGY Glimpses of a Tiny RNA World. SCIENCE VOL 294 26 OCTOBER 2001 Gary Ruvkun  Functions
of microRNAs and related small RNAs in plants.
NATURE GENETICS SUPPLEMENT | VOLUME 38 | JUNE 2006 Allison C Mallory & Hervé Vaucheret MicroRNAs (miRNAs)
and short interfering RNAs (siRNAs), 20- to 27-nt in length, are
essential regulatory molecules that act as sequence-specific
guides in several processes in most eukaryotes (with the notable
exception of the yeast Saccharomyces cerevisiae). These
processes include DNA elimination, heterochromatin assembly, mRNA
cleavage and translational repression. This review focuses
on the regulatory roles of plant miRNAs during development, in the
adaptive response to stresses and in the miRNA pathway itself.
This review also covers the regulatory roles of two classes of
endogenous plant siRNAs, ta-siRNAs and nat-siRNAs, which
participate in post-transcriptional control of gene expression.
Evidence
that microRNAs are associated with translating messenger RNAs in human
cells.
VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY Patricia A Maroney, Yang Yu, Jesse Fisher & Timothy W Nilsen MicroRNAs (miRNAs)
regulate gene expression post-transcriptionally by binding the 3¢
untranslated regions of target mRNAs. We examined the
subcellular distribution of three miRNAs in exponentially growing HeLa
cells and found that the vast majority are associated with
mRNAs in polysomes. Several lines of evidence indicate that most of
these mRNAs, including a known miRNA-regulated
target (KRAS mRNA), are actively being translated.
How
microRNAs control cell division, differentiation and death.
Eric A Miska Current Opinion in Genetics & Development 2005, 15: 563–568 After the milestone
discovery of the first microRNA in 1993, the past five years have
seen a phenomenal surge of interest in these short,
regulatory RNAs. Given that 2% of all known human genes encode
microRNAs, one main goal is to uncover microRNA function.
Although it has been more difficult to assign function to
microRNAs in animals than it has been in plants, important
roles are emerging: in invertebrates, microRNAs control
developmental timing, neuronal differentiation,
tissue growth and programmed cell death. Functional studies in
zebrafish and mice point toward important roles for microRNAs
during morphogenesis and organogenesis.
Finally, microRNAs might regulate viral infection and human
cancer.
microRNA
target predictions in animals.
In recent
years, microRNAs (miRNAs) have emerged as a major class of regulatory
genes, present in most metazoans and important for a
diverse range of biological functions. Because experimental
identification of miRNA targets is difficult, there has been an explosion
of computational target predictions. Although the initial
round of predictions resulted in very diverse results,
subsequent computational and experimental analyses suggested
that at least a certain class of conserved miRNA targets can be
confidently predicted and that this class of targets is
large, covering, for example, at least 30% of all human genes
when considering about 60 conserved vertebrate miRNA gene
families. Most recent approaches have also shown that
there are correlations between domains of miRNA expression
and mRNA levels of their targets. Our understanding of
miRNA function is still extremely limited, but it may be that by
integrating mRNA and miRNA sequence and expression data with
other comparative genomic data, we will be able to gain
global and yet specific insights into the function and evolution of a
broad layer of post-transcriptional control.VOLUME 38 | JUNE 2006 | NATURE GENETICS SUPPLEMENT Nikolaus Rajewsky Human
let-7a miRNA blocks protein production on actively translating
polyribosomes.
MicroRNAs
(miRNAs) regulate gene expression at a post-transcriptional level
through base-pairing to 3¢ untranslated regions (UTRs) of messenger
RNAs. The mechanism by which human let-7a miRNA regulates mRNA
translation was examined in HeLa cells expressing
reporter mRNAs containing the Caenorhabditis elegans lin-41 3¢
UTR. let-7a miRNA strongly repressed translation, yet the
majority of control and lin-41–bearing RNAs sedimented with
polyribosomes in sucrose gradients; these polyribosomes,
together with let-7a miRNA and the miRISC protein AGO, were released
from those structures by puromycin. RNA containing the
lin-41 3¢ UTR and an iron response element in the 5¢ UTR
sedimented with polysomes when cells were incubated with iron,
but showed ribosome run-off when the iron was chelated. These data
indicate that let-7a miRNA inhibits actively translating
polyribosomes. Nascent polypeptide coimmunoprecipitation experiments
further suggest that let-7a miRNA interferes with the
accumulation of growing polypeptides.VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY Stephanie Nottrott, Martin J Simard & Joel D Richter Computational
identification of microRNA targets.
Developmental Biology 267 (2004) 529– 535 Nikolaus Rajewskya, and Nicholas D. Soccib, Department of Biology, New York University, New York, NY 10003-6688, USA; Department of Pathology, and Seaver Foundation for Bioinformatics, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA Recent experiments
have shown that the genomes of organisms such as worm, fly, human, and
mouse encode hundreds of microRNA genes. Many of these
microRNAs are thought to regulate the translational expression of other
genes by binding to partially complementary sites in messenger
RNAs. Phenotypic and expression analysis suggests an important role of
microRNAs during development. Therefore, it is of fundamental
importance to identify microRNA targets. However, no experimental or
computational high-throughput method for target site identification in
animals has been published yet. Our main result is a new computational
method that is designed to identify microRNA target sites. This method
recovers with high specificity known microRNA target sites that have
previously been defined experimentally. Based on these results, we
present a simple model for the mechanism of microRNA target site
recognition. Our model incorporates both kinetic and thermodynamic
components of target recognition. When we applied our method to a set
of 74 Drosophila melanogaster microRNAs, searching 3V UTR
sequences of a predefined set of fly mRNAs for target sites which were
evolutionary conserved between D. melanogaster and Drosophila
pseudoobscura, we found that many key developmental body patterning
genes such as hairy and fushi-tarazu are likely to be translationally
regulated by microRNAs.
Incorporating
structure to predict microRNA targets.
PNAS 2005 vol. 102 no. 11, 4006–4009 Harlan Robins*†, Ying Li‡, and Richard W. Padgett‡ *Institute for Advanced Study, Olden Lane, Princeton, NJ 08540; and ‡Department of Molecular Biology and Biochemistry, Waksman Institute, Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-1020 MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including Caenorhabditis elegans, Drosophila, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. (2001) Science 294, 853–858; Lai, E. C., Tomancak, P., Williams, R. W. & Rubin, G.M. (2003) Genome Biol. 4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. (2001) Science 294, 858–862; Lee, R. C. & Ambros, V. (2001) Science 294, 862-8644; and Lee, R. C., Feinbaum, R. L. & Ambros, V. (1993) Cell 115, 787–798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3 UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using Drosophila miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is mad as a target for bantam. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported. 220-plex microRNA expression profile of a single cell. NATURE PROTOCOLS 2006 VOL.1 NO.3 1154 Fuchou Tang1, Petra Hajkova1, Sheila C Barton1, Do´ nal O’Carroll2, Caroline Lee1, Kaiqin Lao3 & M Azim Surani1 Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d. Nature Letters Vol 448:5 2007 J. Graham Ruby1,2*, Calvin H. Jan1,2* & David P. Bartel1,2 MicroRNAs (miRNAs)
are 22-nucleotide endogenous RNAs that often repress
the expression of complementary messenger RNAs1. In animals,
miRNAs derive from characteristic hairpins in primary
transcripts through two sequential RNase III-mediated cleavages; Drosha
cleaves near the base of the stem to liberate a 60-nucleotide
pre-miRNA hairpin, then Dicer cleaves near the loop to generate a
miRNA:miRNA* duplex2,3. From that duplex, the mature miRNA is
incorporated into the silencing complex. Here we identify an
alternative pathway for miRNA biogenesis, in which certain
debranched introns mimic the structural features of pre-miRNAs to
enter the miRNA-processing pathway without Drosha-mediated
cleavage. We call these pre-miRNAs/introns ‘mirtrons’, and have
identified 14 mirtrons in Drosophila melanogaster and another four in
Caenorhabditis elegans (including the reclassification of
mir-62). Some of these have been selectively maintained during
evolution with patterns of sequence conservation suggesting important
regulatory functions in the animal. The abundance of
introns comparable in size to pre-miRNAs appears to have
created a context favourable for the emergence of mirtrons in flies and
nematodes. This suggests that other lineages with many similarly
sized introns probably also have mirtrons, and that the mirtron
pathway could have provided an early avenue for the emergence of
miRNAs before the advent of Drosha.
 MicroInspector: a web tool for detection of miRNA binding sites in an RNA sequence. Nucleic Acids Research, 2005, Vol. 33, Web Server issue W696–W700 Ventsislav Rusinov1,2, Vesselin Baev1,2, Ivan Nikiforov Minkov2 and Martin Tabler1,* 1Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, PO Box 1527, GR -71110 Heraklion /Crete, Greece and 2 Depar tment of Pla nt Phys iology and Mole cular Biology, Universit y of Plo vdiv 24, Tsar Assen St, 4000 Plovdiv, Bulgaria Regulation
ofpost-transcriptionalgeneexpressionby microRNAs (miRNA) has
so far been validated for only a few mRNA targets.
Based on the large number of miRNA genes and the
possibility that one miRNA might influence gene
expression of several targets simultaneously, the
quantity of ribo-regulated genes is expected to be
much higher. Here, we describe the web tool
MicroInspector that will analyse a userdefined RNA sequence, which
is typically an mRNA or a part of an mRNA,
for the occurrence of binding sites for known and
registered miRNAs. The program allows variation of
temperature, the setting of energy values as well as the
selection of different miRNA databases to identify
miRNA-binding sites of different strength.
MicroInspector could spot the correct sites for miRNA-interaction
in known target mRNAs. Using other mRNAs, for
which such an interaction has not yet been described,
we discovered frequently potential miRNA binding sites
of similar quality, which can now be analysed
experimentally. The MicroInspector program is easy to
use and does not require specific computer skills. The
service can be accessed via the MicroInspector
web server at http://www.imbb.forth.gr/microinspector
Target labelling for
the detection and profiling of microRNAs expressed in CNS tissue using
microarrays.BMC Biotechnology 2006, 6:47 Reuben Saba1,2 and Stephanie A Booth*1,2 1Division of Host Genetics & Prion Diseases, National Microbiology Laboratory, 1015 Arlington Street, Public Health Agency of Canada, Winnipeg, MB, R3E 3R2, Canada and 2Department of Medical Microbiology and Infectious Diseases, Faculty of Medicine, University of Manitoba, Winnipeg, MB, R3B 1Y6, Canada Background: MicroRNAs
(miRNA) are a novel class of small, non-coding, gene regulatory RNA
molecules that have diverse roles in a variety of eukaryotic biological
processes. High-throughput detection and differential expression
analysis of these molecules, by microarray technology, may contribute
to a greater understanding of the many biological events regulated by
these molecules. In this investigation we compared two different
methodologies for the preparation of labelled miRNAs from mouse CNS
tissue for microarray analysis. Labelled miRNAs were prepared either by
a procedure involving linear amplification of miRNAs (labelled-aRNA) or
using a direct labelling strategy (labelled-cDNA) and analysed using a
custom miRNA microarray platform. Our aim was to develop a rapid,
sensitive methodology to profile miRNAs that could be adapted for use
on limited amounts of tissue. Results: We demonstrate the detection of
an equivalent set of miRNAs from mouse CNS tissues using both amplified
and non-amplified labelled miRNAs. Validation of the expression of
these miRNAs in the CNS by multiplex real-time PCR confirmed the
reliability of our microarray platform. We found that although the
amplification step increased the sensitivity of detection of miRNAs, we
observed a concomitant decrease in specificity for closely related
probes, as well as increased variation introduced by dye bias.
Conclusion: The data presented in this investigation identifies several
important sources of systematic bias that must be considered upon
linear amplification of miRNA for microarray analysis in comparison to
directly labelled miRNA.
A brain-specific microRNA regulates dendritic spine development. Gerhard M. Schratt1,2,3, Fabian Tuebing4, Elizabeth A. Nigh1,2,3, Christina G. Kane1,2,3, Mary E. Sabatini3, Michael Kiebler4 & Michael E. Greenberg1,2,3 Nature Vol 439:19 January 2006 MicroRNAs are small, non-coding RNAs that control the translation of target messenger RNAs, thereby regulating critical aspects of plant and animal development. In the mammalian nervous system, the spatiotemporal control of mRNA translation has an important role in synaptic development and plasticity. Although a number of microRNAs have been isolated from the mammalian brain, neither the specific microRNAs that regulate synapse function nor their target mRNAs have been identified. Here we show that a brain-specific microRNA, miR-134, is localized to the synaptodendritic compartment of rat hippocampal neurons and negatively regulates the size of dendritic spines-postsynaptic sites of excitatory synaptic transmission. This effect is mediated by miR-134 inhibition of the translation of an mRNA encoding a protein kinase, Limk1, that controls spine development. Exposure of neurons to extracellular stimuli such as brain-derived neurotrophic factor relieves miR-134 inhibition of Limk1 translation and in this way may contribute to synaptic development, maturation and/or plasticity. Oncogene (2006), 1–5 M-L Si, S Zhu, H Wu, Z Lu, F Wu and Y-Y Mo Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, USA MicroRNAs (miRNAs)
are B22 nucleotide non-coding RNA molecules that
regulate gene expression post-transcriptionally. Although aberrant
expression of miRNAs in various human cancers
suggests a role for miRNAs in tumorigenesis, it
remains largely unclear as to whether knockdown of a
specific miRNA affects tumor growth. In this study, we
profiled miRNA expression in matched normal breast tissue and
breast tumor tissues by TaqMan real-time polymerase chain
reaction miRNA array methods. Consistent with previous
findings, we found that miR-21 was highly overexpressed in
breast tumors compared to the matched normal breast tissues
among 157 human miRNAs analysed. To better evaluate
the role of miR-21 in tumorigenesis, we
transfected breast cancer MCF-7 cells with anti-miR-21 oligonucleotides and found that anti-miR-21 suppressed both cell growth in vitro and tumor growth in the xenograft mouse model. Furthermore, this anti-miR-21-mediated cell growth inhibition was associated with increased apoptosis and decreased cell proliferation, which could be in part owing to downregulation of the antiapoptotic Bcl-2 in anti-miR-21- treated tumor cells. Together, these results suggest that miR-21 functions as an oncogene and modulates tumorigenesis through regulation of genes such as bcl-2 and thus, it may serve as a novel therapeutic target.  |