strand specific sequencing of RNAs from MAoECs to determine the endothelial-specific expression profile of protein-coding and long non-coding RNAs Overall design: Total RNA was isolated from cultured MAoECs (passage 4) and processed for a strand-specific RNA sequencing. The RNA purity and integrity were assessed using the Fragment Analyzer Automated CE System (Advanced Analytical). A RQN of 8.8 and a 28S/18S ratio of 2.2 were considered acceptable for next generation sequencing assay. Five µg of DNase-treated RNA were used to prepare Massive Analysis of cDNA ends (MACE) libraries needed to perform a DNA-Methylation-Sequencing (Meth-Seq) PCR bias free quantification with TrueQuant Technology, followed by a high-throughput sequencing on the Illumina Genome Analyzer II system (GenXPro GmbH, Frankfurt, Germany). The procedure consist in the extraction of poly-adenylated RNA from 5 µg RNA and reverse transcribed with biotinylated poly(T) primers. cDNA is fragmented to an average size of 250 bp. Biotinylated ends are captured by streptavidin beads and ligated to modified adapters (TrueQuant DNA adapter, GenXPro). The libraries are amplified by PCR, purified by SPRI beads and sequenced (2 x 100 bp Illumina HiSeq2000 TrueSeq, 2 x 20 Mio. Reads poly-A selected paired-end reads). Paired end sequencing of both DNA strands from each end is required for fragment strand specificity.
miR-103 promotes endothelial maladaptation by targeting lncWDR59.
Specimen part, Subject
View SamplesPurpose: The goal of the present study is to provide an independent assessment of the retinal transcriptome signatures of the C57BL/6J (B6) and DBA/2J (D2) mice and to enhance existing microarray datasets for accurately defining the allelic differences in the BXD recombinant inbred strains. Methods: Retinas from both B6 and D2 mice (3 of each) were used for the RNA-seq analysis. Transcriptome features were examined for both strains. Differentially expressed genes between the 2 strains were identified and bioinformatic analysis was performed to analyze the transcriptome differences between B6 and D2 strains, including Gene ontology (GO) analysis, Phenotype and Reactome enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The RNA-seq data were then directly compared with one of the microarray datasets (DoD Retina Normal Affy MoGene 2.0 ST RMA Gene Level Microarray Database) hosted on GeneNetwork (www.genenetwork.org). Results: RNA-seq provided an in-depth analysis of the transcriptome of the B6 and D2 retina with a total of more than 30,000,000 reads per sample. Over 70% of the reads were uniquely mapped, resulting in a total of 18,100 gene counts for all 6 samples. 1,665 genes were differentially expressed, with 858 of these more highly expressed in B6 and 807 more highly expressed in D2. Several molecular pathways were differentially active between the two strains, including the retinoic acid metabolic process, endoplasmic reticulum lumen, extracellular matrix organization, and PI3K-Akt signaling pathway. The most enriched KEGG pathways were the pentose and glucuronate interconversions pathway, the cytochrome P450 pathway, protein digestion and absorption pathway and the ECM-receptor interaction pathway. Each of these pathways had a more than 4-fold enrichment. The DoD normal retina microarray database provided expression profiling for 26,191 annotated transcripts for B6 mouse, D2 mouse and 53 BXD strains. A total of 13,793 genes in this microarray dataset were comparable to the RNA-seq dataset. For both B6 and D2, the RNA-seq data and microarray data were highly correlated with each other (Pearson's r = 0.780 for B6 and 0.784 for D2). Our results suggest that the microarray dataset can reliably detect differentially expressed genes between the B6 and D2 retinas, with a positive predictive value of 45.6%, and a negative predictive value of 93.6%. Examples of true positive and false positive genes are provided. Conclusions: Retinal transcriptome features of B6 and D2 mouse strains provide a useful reference for a better understanding of the mouse retina. Generally, the microarray database presented on GeneNetwork shows good agreement with the RNA-seq data, while we note that any allelic difference between B6 and D2 should be verified with the latter. Overall design: Retinal mRNA profiles of 2 strains of mice, C57BL/6J and DBA/2J, were generated by deep sequencing, in triplicate, using Illumina TruSeq Stranded Total RNA kit.
RNA sequencing profiling of the retina in C57BL/6J and DBA/2J mice: Enhancing the retinal microarray data sets from GeneNetwork.
Specimen part, Cell line, Subject
View SamplesAmputation of heart tissue followed by regeneration of the heart. Samples were taken at 0 hpa (hours post-amputation), 6 hpa, 12 hpa, 24 hpa, 3 dpa and 5 dpa.
Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration.
Specimen part, Time
View SamplesThe objective of the present investigation was to utilize the GeneChip Porcine Genome Array from Affymetrix possessing 20, 201 unique probe sets to identify differentially expressed genes during rapid trophoblastic elongation and attachment to the uterine surface in the pig. Identification and characterization of conceptus gene expression patterns during rapid trophoblastic elongation and attachment in the pig will provide a better understanding of the events required for successful implantation and embryonic survival.
Identification of differential gene expression during porcine conceptus rapid trophoblastic elongation and attachment to uterine luminal epithelium.
No sample metadata fields
View SamplesPlacentation of the conceptus to the surface epithelium is governed through a tightly regulated temporal and spatial window. Premature exogenous steriod exposure causes a shift in the maternal tissue's receptivity and prevents proper placentation.
Effects of aberrant estrogen on the endometrial transcriptional profile in pigs.
Specimen part
View SamplesWe used NEBNext Ultra Directional RNA Library Prep Kits to prepare RNA-seq libraries of total RNA from hnRNP A2/B1 and A1 depleted A549 cells. Pro-seq libraries were prepared from A549 cells using Illumina adapters Overall design: hnRNP A2/B1 and A1 depleted A549 cells were generated by lentiviral infections of shRNA constructs. RNAs were isolated using Trizol.
A widespread sequence-specific mRNA decay pathway mediated by hnRNPs A1 and A2/B1.
No sample metadata fields
View SamplesInterstitial cystitis (IC), a chronic bladder disease with an increasing incidence, is diagnosed using subjective symptoms in combination with cystoscopic and histological evidence. The ultimate goal is the development of a diagnostic assay for IC on a molecular level.
Gene expression profile of bladder tissue of patients with ulcerative interstitial cystitis.
Specimen part, Disease, Disease stage
View Samples10 days old tumor spheroids were processed for RNA isolation using the Quiagen RNeasy Micro Kit and cDNA synthesis was done using the Ambion WT Expression Kit. Wt, HIF-1 k/d and HIF-2 k/d samples were compared to each other.
HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma.
Specimen part, Cell line
View SamplesWe performed RNA-seq analysis of WT and blmp-1(tm548) mutant L3 larvae to identify genes regulated by the zing-finger transcription factor BLMP-1. Overall design: We analyzed three WT and three blmp-1 mutant biological replicates
DRE-1/FBXO11-dependent degradation of BLMP-1/BLIMP-1 governs C. elegans developmental timing and maturation.
Cell line, Subject
View SamplesTo better understand the pathogenesis of acute promyelocytic leukemia (APL, FAB M3 AML), we identified genes that are expressed differently in APL cells compared to other acute myeloid leukemia subtypes, and to normal promyelocytes. Comparative gene expression analysis of 14 M3, 62 other AML (M0, M1, M2 and M4) and 5 enriched normal promyelocyte samples revealed a signature of 1,121 genes that are specifically dysregulated in M3 samples relative to other AML, and that do not simply represent normal promyelocyte expression (M3-specific signature). We used a novel, high throughput digital platform (Nanostring's nCounter system) to evaluate a subset of the most significantly dysregulated genes in 30 AML samples; 33 of 37 evaluable gene expression patterns were validated. In an additional analysis, we selected only genes that are dysregulated in M3 both compared to other AML subtypes, and to purified normal CD34+ cells, promyelocytes, and/or neutrophils, thereby isolating a 478 gene "composite M3 dysregulome". Surprisingly, the expression of only a few of these genes was significantly altered in PR-9 cells after PML-RARA induction, suggesting that most of these genes are not direct targets of PML-RARA. Comparison of the M3-specific signature to our previously described murine APL dysregulome revealed 33 commonly dysregulated genes, including JUN, EGR1, and TNF. Collectively, these results suggest that PML-RARA initiates a transcriptional cascade which generates a unique downstream expression signature in both primary human and mouse APL cells.
High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples.
Sex, Race
View Samples