Reverse transcription is the first step of most analyses of gene expression, yet the quantitative biases it introduces are largely overlooked. Following a series of purpose-designed systematic ...experiments we cherry-pick examples of various biases introduced by reverse transcription, and alert the "gene expression community" to the pitfalls and improved practice of this fundamental technique.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
RNA abundance and DNA copy number are routinely measured in high-throughput using microarray and next-generation sequencing (NGS) technologies, and the attributes of different platforms have been ...extensively analyzed. Recently, the application of both microarrays and NGS has expanded to include microRNAs (miRNAs), but the relative performance of these methods has not been rigorously characterized. We analyzed three biological samples across six miRNA microarray platforms and compared their hybridization performance. We examined the utility of these platforms, as well as NGS, for the detection of differentially expressed miRNAs. We then validated the results for 89 miRNAs by real-time RT-PCR and challenged the use of this assay as a "gold standard." Finally, we implemented a novel method to evaluate false-positive and false-negative rates for all methods in the absence of a reference method.
MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast ...tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA-mRNA interactions rather than as on-off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite their abundance, mid-sized RNAs (30–300 nt) have not been extensively studied by high-throughput sequencing, mostly due to selective loss in library preparation. The authors propose simple ...and inexpensive modifications to the Illumina TruSeq protocol (ncRNAseq), allowing the capture and sequencing of mid-sized non-coding RNAs without detriment to the coverage of coding mRNAs. This protocol is coupled with a two-step alignment: a pre-alignment to a curated non-coding genome, passing only the non-mapping reads to a standard genomic alignment. ncRNAseq correctly assigns the highest read-numbers to established abundant non-coding RNAs and correctly identifies cytosolic and nuclear enrichment of known non-coding RNAs in two cell lines.
In order to retain RNAs shorter than 300 nt and the cDNAs they give rise to, the authors reduce the fractionation time of the RNA and increase the efficiency of four precipitation steps in the Illumina TruSeq protocol (ncRNAseq): twice pre-ligation by adding isopropanol to AMPure beads and twice post-ligation by using a higher AMPure-to-sample ratio. The resulting sequencing reads are aligned first to a custom non-redundant curated ‘non-coding genome’, which allows the user to exert control, for example, on amalgamation of multi-copy genes or separation of non-coding intronic gene counts from coding host gene counts. The non-mapping reads are then passed to a standard genomic alignment in a lossless manner.
MicroRNAs are short (17-26) noncoding RNAs driving or modulating physiological and pathological cellular events. Overexpression of miR-155 is pathogenic in B-cell malignancy but was also reported in ...a number of solid tumors-in particular, in breast cancer, where its role remains unclear and often contradictory. Using representative cell line models, we sought to determine whether the discrepant miR-155 effects in breast cancer could be explained by the heterogeneity of the disease. The growth of six breast cancer cell lines transfected with several miRNA mimics was analyzed. We found MCF-7 cell growth to be inhibited by miR-155 and miR-145 mimics, both 23-nt long, but not by a number of shorter mimics, including a universal commercial negative control. Microarray and Western blot analyses revealed induction of apoptosis, associated with interferon-β after activation of the double-stranded RNA sensor pathway. 3' Trimming of the miRNA mimics to 21 nt substantially reduced their growth-inhibitory potency. Mutating the canonical seed of the miR-155 mimic had no effect on the induced inhibition, which was abolished by mutating the miRNA seed of the artificial passenger strand. A panel of breast cancer cell lines showed a wide range of sensitivities to 23-mer mimics, broadly consistent with the sensitivity of the cell lines to Poly (I:C). We demonstrate two sources for nonspecific in vitro effects by miRNA mimics: duplex length and the artificial passenger strand. We highlight the danger of a universal 21-mer negative control and the importance of using matched seed mutants for reliable interpretation of phenotypes.
Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the ...translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3'UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3'UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Amplification of the EMSY gene in sporadic breast and ovarian cancers is a poor prognostic indicator. Although EMSY has been linked to transcriptional silencing, its mechanism of action is unknown. ...Here, we report that EMSY acts as an oncogene, causing the transformation of cells in vitro and potentiating tumor formation and metastatic features in vivo. We identify an inverse correlation between EMSY amplification and miR-31 expression, an antimetastatic microRNA, in the METABRIC cohort of human breast samples. Re-expression of miR-31 profoundly reduced cell migration, invasion, and colony-formation abilities of cells overexpressing EMSY or haboring EMSY amplification. We show that EMSY is recruited to the miR-31 promoter by the DNA binding factor ETS-1, and it represses miR-31 transcription by delivering the H3K4me3 demethylase JARID1b/PLU-1/KDM5B. Altogether, these results suggest a pathway underlying the role of EMSY in breast cancer and uncover potential diagnostic and therapeutic targets in sporadic breast cancer.
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•EMSY is an oncogene that represses miR-31 transcription in a BRCA2-independent manner•Re-expression of miR-31 abrogates EMSY-mediated effects on cell migration in vitro•EMSY and miR-31 levels anticorrelate in human breast samples from the METABRIC cohort•EMSY, ETS-1, and KDM5B co-occupy miR-31 promoter and repress its transcription
EMSY gene amplification is an indicator of breast and ovarian cancers. Viré et al. report that EMSY is an oncogene that functions by repressing the transcription of antimetastatic miR-31. Repression involves the transcription factor ETS-1 and the histone demethylase KDM5B. miR-31 is a key player in cancer-related EMSY function because miR-31 expression reduces EMSY-mediated oncogenicity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Group-2 innate lymphoid cells (ILC2) play critical roles in the initiation and maintenance of type-2 immune responses, predominantly through their production of the type-2 cytokines IL-5, IL-9, and ...IL-13. ILC2 are essential for the efficient elimination of helminth parasites, but also contribute to the detrimental type-2 immune responses that underlie diseases such as asthma and allergy. While several transcription factors have been identified that regulate the development and function of ILC2, less is known about the post-transcriptional mechanisms that regulate these processes. We identified micro-RNAs (miRNAs) that are co-ordinately regulated in ILC2 from mice exposed to two different stimuli, namely IL-33 "alarmin" administration or
parasitic worm infection. miR-155 is upregulated in ILC2 in response to both stimuli and miR-155
mice had impaired IL-33-driven ILC2 responses. Using mixed bone marrow chimeras, we demonstrate that this deficit is intrinsic to ILC2 and that miR-155 protects ILC2 from apoptosis, while having little impact on ILC2 proliferation or cytokine production. These data reveal a subset of miRNAs that are regulated upon ILC2 activation and establish a specific role for miR-155 in regulating ILC2 survival following activation.
A recipe for disaster Git, Anna
Nature,
04/2012, Volume:
484, Issue:
7395
Journal Article
Peer reviewed
Open access
The sequence was too short, leading to results that were impossible to interpret, if not just wrong. Because the company didn't reveal much information about the product, we only discovered the ...discrepancy fortuitously after testing many microRNAs of known sequence, and observing a length-dependent activity among them. Reviewers of papers should also require this information. Because of the too-short sequence my student used as a negative control, one-quarter of her PhD was wasted.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK