Cross platform standardization of an experimental pipeline for use in the identification of dysregulated human circulating miRNAs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Tim Downing
PROVIDER: E-GEOD-72080 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The precise control of miR-17~92 microRNA (miRNA) is essential for normal development and overexpression of certain miRNAs from this cluster is oncogenic. Here we find the relative expression of the six miRNAs processed from the primary (pri-miR-17~92) transcript is dynamically regulated during embryonic stem cell differentiation. We identify a new miRNA biogenesis intermediate, termed ‘progenitor-miRNA’ (pro-miRNA), that is an efficient substrate for Microprocessor. An autoinhibitory 5’ RNA fragment is cleaved to generate pro-miRNA and selectively license Microprocessor-mediated production of pre-miR-17, -18a, -19a, 20a, and -19b. Using genetic, biochemical, and structural methods we define two complementary cis-regulatory repression domains required for the formation of this inhibitory RNA conformation. We find the endonuclease CPSF3 (CPSF73), and the Spliceosome-associated ISY1 are required for pro-miRNA biogenesis and expression of all miRNAs within the cluster except miR-92. Thus, developmentally regulated generation of pro-miRNA explains the posttranscriptional control of miR-17~92 expression in development. Illumina RNAseq in WT, dgcr8-/- and dicer-/- mESCs and small RNA seq in WT mESCs
2015-05-11 | E-GEOD-68699 | biostudies-arrayexpress
Project description:In this study we present an experimental pipeline that takes into consideration sample collection, processing, enrichment, and the subsequent comparative analysis of circulating small ribonucleic acids using small RNA sequencing and RT-qPCR. Initially, a panel of miRNAs dysregulated in circulating blood from breast cancer patients compared to healthy women were identified using small RNA sequencing. MiR-320a was identified as the most dysregulated miRNA between the two female cohorts. Total RNA and enriched small RNA populations (<30 bp) isolated from peripheral blood from the same female cohort samples were then tested using a miR-320a RT-qPCR assay. When total RNA was analyzed with this miR-320a RT-qPCR assay, a 2.3-fold decrease in expression levels was observed between blood samples from healthy controls and breast cancer patients. However, upon enrichment for the small RNA population and subsequent analysis of miR-320a using RT-qPCR, its dysregulation in breast cancer patients was more pronounced with an 8.89-fold decrease in miR-320a expression.
2015-08-30 | GSE72080 | GEO
Project description:Background: WitchesM-bM-^@M-^Y broom disease of Mexican lime (Citrus aurantifolia L.), which is caused by the phytoplasma M-bM-^@M-^\Candidatus Phytoplasma aurantifoliaM-bM-^@M-^], is a devastating disease that results in significant economic losses. Plants adapt to abiotic stresses by regulating gene expression at the transcriptional and post-transcriptional levels. MicroRNAs (miRNAs) are a recently identified family of molecules that regulate plant responses to environmental stresses through post-transcriptional gene silencing. Methods: Using a high-throughput approach to sequence small RNAs, we compared the expression profiles of miRNAs in healthy Mexican lime trees and in plants infected with M-bM-^@M-^\Ca. Phytoplasma aurantifoliaM-bM-^@M-^]. Results: Our results demonstrated the involvement of different miRNAs in the response of Mexican lime trees to infection by M-bM-^@M-^\Ca. Phytoplasma aurantifoliaM-bM-^@M-^]. We identified miRNA families that are expressed differentially upon infection with phytoplasmas. Most of the miRNAs had variants with small sequence variations (isomiRs), which are expressed differentially in response to pathogen infection. Conclusions: It is likely that the miRNAs that are expressed differentially in healthy and phytoplasma-infected Mexican lime trees are involved in coordinating the regulation of hormonal, nutritional, and stress signalling pathways, and the complex interactions between them. Future research to elucidate the roles of these miRNAs should improve our understanding of the level of diversity of specific plant responses to phytoplasmas. Small mRNA profiles of healthy (H) and Phytoplasma-infected Mexican lime trees were generated by deep sequencing, six replicate, using Illumina Hiseq2000
2013-03-09 | E-GEOD-44279 | biostudies-arrayexpress
Project description:Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. microRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a by-product of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway. These were independently processed and sequenced using the Illumina GAII platform. In total, five libraries were analyzed.
2009-11-15 | E-GEOD-17734 | biostudies-arrayexpress
Project description:MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length. Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance in the Vitis genus and is used as an excellent breeding parent for grapevine, and with growing interest in terms of wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. In this study, a small RNA library from Amur grapes was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNA belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grapevine-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, accumulation of 18 new va-miRNAs in seven tissues of grapevines were also confirmed by real time RT-PCR (qRT-PCR) analysis, and expression levels of va-miRNAs in flowers and berries were basically consistent in identity to those from deep sequenced sRNAs libraries of independent corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and revealed the number and sites of miR-SNP of diverse miRNA families exhibited distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grapevine stress tolerance genes and many genes regulating anthocyanin systhesis and sugar metabolism. Deep sequencing of short RNAs from Amur grapes flowers and fruits identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grapes. These results show that a number of regulatory miRNAs exist in Amur grapes and play an important role in Amur grape growth, development, and response to abiotic or biotic stress. High throughput sequencing was employed to identify miRNAs in Amur grapevine and try to describe their functions in Amur grapevine growth and development.
2011-12-16 | E-GEOD-34169 | biostudies-arrayexpress
Project description:Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26-nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the argonaute ERGO-1, DICER, and a series of associated (ERI) factors. This primary process leads to production of a much more abundant class of 22-nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely-structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA. 24 small RNA and 2 polyA RNA samples
2010-02-01 | E-GEOD-19414 | biostudies-arrayexpress
Project description:Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing, raising the interesting possibility for a hidden layer of widespread virus-host interactions that may contribute to viral pathogenicity and host specificity. Profiling of TMV-Cg derived small RNAs in systemically infected tissues of wild type (Col-0) Arabidopsis as well as the rdr1and rdr6 mutants, at 3 days post-infection.
2009-03-24 | E-GEOD-12918 | biostudies-arrayexpress
Project description:The Arabidopsis ARGONAUTE (AGO) protein AGO1 associates with microRNA (miRNA) and specific classes of short-interfering RNA (siRNA). AGO1-small RNA complexes recognize target RNA transcripts through base-pairing interactions and inhibit translation of target RNAs through endonucleolytic cleavage (slicing) or non-degradative mechanisms. The PIWI domain of AGO1 contains a metal-coordinating triad [Asp-Asp-His] (DDH) that is required for slicer activity. Here, we compared the activities of wild type (DDH) and slicer active-site defective (DAH) forms of AGO1 by sequencing small RNA and target transcript RNAs that co-immunoprecipitated with hemagglutinin (HA)-tagged AGO1 proteins. We found that the population of miRNA that associated with both AGO1-DDH and AGO1-DAH proteins largely overlapped, suggesting that cleavage activity does not affect miRNA maturation. In contrast, slicer-defective AGO1-miRNA complexes associated with target RNA more effectively than did wild type AGO1-miRNA. These data indicate that slicer-defective AGO proteins can be used as an approach to capture AGO-small RNA-target RNA ternary complexes more efficiently for genome-wide analyses. AGO1-DDH (wild type) AGO1-DAH (slicer mutant) proteins were immunoprecipitated (N-terminal 3xHA) from Arabidopsis (Columbia) flower (stages 1-12) lysate. Immunoprecipitations were also done from control plants transformed with vector only. Small RNA from immunoprecipitate fractions of vector, AGO1-DDH and AGO1-DAH were sequenced.
2012-09-28 | E-GEOD-40258 | biostudies-arrayexpress
Project description:Endogenous 24nt short interfering RNAs (siRNAs) derived mostly from intergenic and repetitive genomic regions constitute a major class of endogenous small RNAs in Arabidopsis thaliana. Accumulation of A. thaliana 24nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21nt microRNAs (miRNAs) and 21-22nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci which consistently produced a mixture of 21-24nt siRNAs with a peak at 23nts. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22-24nt siRNAs, but not 21nt siRNAs, at Pp23SR loci. The 21nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants specifically failed to accumulate 23 and 24nt small RNAs from repetitive regions. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants. Our results also suggest that Pp DCL3 produces siRNAs of heterogenous size, unlike its A. thaliana homolog which generates exclusively 24nt siRNAs. Small RNAs from Wild-type (two technical replicates), rdr6-19 (two technical replicates), dcl3-5 (one sample), and dcl3-10 (one sample) were sequenced using a Solexa GA. Three different linkers were used for different samples and mixed prior to sequencing. linker 1: CTGTAGGCACCATCAAT (GPL7174 AND GSM313212, GSM313213) linker 2: CACTCGGGCACCAAGGA (GPL7175 AND GSM313214, GSM313215) linker 3: TTTAACCGCGAATTCCAG (GPL7176 AND GSM313216, GSM313217) Raw data: Two FASTQ files represent the raw data underlying the processed sample data in GSE12468. 080616_s_7_seq_GAK-2.fastq corresponds to GSM313212, GSM313214, and GSM313216. 080616_s_8_seq_GAK-3.fastq corresponds to GSM313213, GSM313215, and GSM313217. Experimentally, samples GSM313212, GSM313214, and GSM313216 (i.e., all three genotypes) were sequenced in the same run (hence the same FASTQ file). The sequencing run was a mixture of three different libraries with different linker sequences (as indicated above). These 'barcoded' samples were computationally extracted based on the linker sequences. This is also true for samples GSM313213, GSM313215, and GSM313217. Raw data were filtered to identify small RNAs associated with each linker. Subsequently, small RNAs were filtered to remove rRNA fragments. The remainder were mapped to the Physcomitrella patens draft genome version 1.2 (using oligomap) and filtered to retain only those with one or more perfect match. See Cho et al. for details.
2008-12-19 | E-GEOD-12468 | biostudies-arrayexpress
Project description:MicroRNAs (miRNA), discovered in C. elegans, are short non-coding RNAs that bind and regulate the expression of target mRNAs in animals and plants. C. elegans miRNAs bind to partially complementary sequences in the 3' UTR of the target mRNA, which results in translational repression through mRNA destabilization. The high-throughput sequencing of RNA cleavage fragments was performed to directly detect cleaved miRNA targets in C. elegans. Based on this analysis, miR-249 was identified as a potential miRNA that regulates a ZK637.6 pseudogene, paralogous to asna-1 (ZK637.5), by a cleavage mechanism with extensive, evolutionary conserved complementarity. Additionally, we validated miR-249-directed cleavage of the ZK637.6 by a gene-specific 5M-bM-^@M-^Y rapid amplification of cDNA ends and observed notable difference in expression of ZK637.6 in wild-type versus mir-249 mutant C. elegans. Furthermore, phosphate-independent small-RNA sequencing analysis revealed that miR-249 target genes, including ZK637.6 pseudogene, showed 22G-RNA productions dependent on miR-249 targeting. These findings may lead to a better understanding of the biological roles of miRNAs for pseudogenes in C. elegans. Total small RNAs from wild-type and mir-249 mutant in adult stage worms were subjected to small RNA sequencing using an Illumina platform with Tobacco Acid Pyrophosphatase (TAP) treatment, allowing detection of secondary siRNAs carrying 5M-bM-^@M-^Y-tri-phosphate.
2013-08-08 | E-GEOD-41900 | biostudies-arrayexpress