Identification of the 3' end of the pig miRNA by the RAKE technology [platform 2: 9003179]
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ABSTRACT:
ORGANISM(S): Sus scrofa
PROVIDER: GSE28138 | GEO | 2014/03/31
SECONDARY ACCESSION(S): PRJNA143153
REPOSITORIES: GEO
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Project description:We applied a new computational approach to predict specie-specific and conserved miRNAs, than experimentally confirmed by a modified RNA-primed Array-based Klenow Extension (RAKE) method. We identified 489 conserved and 1,178 pig-specific novel miRNAs increasing our tally of confirmed miRNAs to 1,667 novel miRNAs. In addition, RAKE allowed the identification of miRNA isoforms (isomiRs) that we demonstrated to be differentially expressed across tissues suggesting that subtle variability in isomiR expression is regulated and biologically meaningful.
2014-03-31 | GSE28139 | GEO
Project description:We applied a new computational approach to predict specie-specific and conserved miRNAs, than experimentally confirmed by a modified RNA-primed Array-based Klenow Extension (RAKE) method. We identified 489 conserved and 1,178 pig-specific novel miRNAs increasing our tally of confirmed miRNAs to 1,667 novel miRNAs. In addition, RAKE allowed the identification of miRNA isoforms (isomiRs) that we demonstrated to be differentially expressed across tissues suggesting that subtle variability in isomiR expression is regulated and biologically meaningful.
2014-03-31 | GSE28137 | GEO
Project description:We applied a new computational approach to predict specie-specific and conserved miRNAs, than experimentally confirmed by a modified RNA-primed Array-based Klenow Extension (RAKE) method. We identified 489 conserved and 1,178 pig-specific novel miRNAs increasing our tally of confirmed miRNAs to 1,667 novel miRNAs. In addition, RAKE allowed the identification of miRNA isoforms (isomiRs) that we demonstrated to be differentially expressed across tissues suggesting that subtle variability in isomiR expression is regulated and biologically meaningful. 12K microarray was hybridized with a pool of small RNAs from 20 different tissues (superior vena cava, adipose tissue, lung, spleen, stomach, liver, intestine, kidney, descending aorta, left atrium, left ventricle, skeletal muscle, pulmonary aorta, skin, tongue, ascending aorta, arterial white cells blood, venal white cells blood, coronary valve, lymph node). Same experiment was replicated three times to grant the reliability of the identified 5M-bM-^@M-^Y end of miRNAs and microarray slides were scanned three times at low, medium and high PMT to improve microarray dynamic range.
2014-03-31 | E-GEOD-28139 | biostudies-arrayexpress
Project description:We applied a new computational approach to predict specie-specific and conserved miRNAs, than experimentally confirmed by a modified RNA-primed Array-based Klenow Extension (RAKE) method. We identified 489 conserved and 1,178 pig-specific novel miRNAs increasing our tally of confirmed miRNAs to 1,667 novel miRNAs. In addition, RAKE allowed the identification of miRNA isoforms (isomiRs) that we demonstrated to be differentially expressed across tissues suggesting that subtle variability in isomiR expression is regulated and biologically meaningful. 90K microarray was hybridized with a pool of small RNAs from 20 different tissues (superior vena cava, adipose tissue, lung, spleen, stomach, liver, intestine, kidney, descending aorta, left atrium, left ventricle, skeletal muscle, pulmonary aorta, skin, tongue, ascending aorta, arterial white cells blood, venal white cells blood, coronary valve, lymph node). Same experiment was replicated four times to grant the reliability of the identified 3M-bM-^@M-^Y end of miRNAs and microarray slides were scanned three times at low, medium and high PMT to improve microarray dynamic range.
2014-03-31 | E-GEOD-28137 | biostudies-arrayexpress
Project description:We applied a new computational approach to predict specie-specific and conserved miRNAs, than experimentally confirmed by a modified RNA-primed Array-based Klenow Extension (RAKE) method. We identified 489 conserved and 1,178 pig-specific novel miRNAs increasing our tally of confirmed miRNAs to 1,667 novel miRNAs. In addition, RAKE allowed the identification of miRNA isoforms (isomiRs) that we demonstrated to be differentially expressed across tissues suggesting that subtle variability in isomiR expression is regulated and biologically meaningful. 90K microarray was hybridized with a pool of small RNAs from 20 different tissues (superior vena cava, adipose tissue, lung, spleen, stomach, liver, intestine, kidney, descending aorta, left atrium, left ventricle, skeletal muscle, pulmonary aorta, skin, tongue, ascending aorta, arterial white cells blood, venal white cells blood, coronary valve, lymph node). Same experiment was replicated four times to grant the reliability of the identified 3M-bM-^@M-^Y end of miRNAs and microarray slides were scanned three times at low, medium and high PMT to improve microarray dynamic range.
2014-03-31 | E-GEOD-28138 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are small regulatory RNAs, which serve fundamental biological roles across eukaryotic species. We describe a novel method for high throughput miRNA detection. The technique is termed RNA-primed, Array-based, Klenow Enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over Northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalinfixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling. Keywords = microRNA Keywords: ordered
2004-11-01 | GSE1821 | GEO
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
2014-08-06 | E-GEOD-58734 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are small regulatory RNAs, which serve fundamental biological roles across eukaryotic species. We describe a novel method for high throughput miRNA detection. The technique is termed RNA-primed, Array-based, Klenow Enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over Northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalinfixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling. Keywords = microRNA Keywords: ordered
2010-06-05 | E-GEOD-1821 | biostudies-arrayexpress
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
2014-08-06 | GSE58734 | GEO
Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10-18 moles to 10-14 moles in a linear range) (see Figure), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 14 different tissues relating it with tissue anatomical proximity and functional similarity.
2014-03-31 | GSE28140 | GEO