We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen.
Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes.
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View SamplesSle1c is a sublocus of the NZM2410-derived Sle1 major susceptibility locus. We have previously shown that Sle1c contributes to lupus pathogenesis by conferring CD4+ T cell-intrinsic hyperactivation and increased susceptibility to chronic graft-versus-host disease (cGVHD) that mapped to the centromeric portion of the locus. In this study, we have refined the centromeric sublocus to a 675Kb interval, termed Sle1c2. Recombinant congenic strains expressing Sle1c2 exhibited a T cell-intrinsic CD4+ T cell hyperactivation and cGVHD susceptibility, similar to mice with the parental Sle1c.
Murine lupus susceptibility locus Sle1c2 mediates CD4+ T cell activation and maps to estrogen-related receptor γ.
Sex, Age, Specimen part
View SamplesThe goal of our study was to evaluate at the systems-level, the effect of sex hormones on thymic epithelial cells (TECs). To this end, we sequenced the transcriptome of cortical and medullary TECs (cTECs and mTECs) from three groups of 6 month-old mice: males, females and males castrated at four weeks of age. In parallel, we analyzed variations in the size of TEC subsets in those three groups between 1 and 12 months of age. We report that sex hormones have pervasive effects on the transcriptome of TECs: the number of differentially expressed genes was 1,440 in cTECs and 1,783 in mTECs. Sexual dimorphism was particularly conspicuous in cTECs. Male cTECs displayed low proliferation rates that correlated with low expression of Foxn1 and its main targets. Furthermore, male cTECs expressed relatively low levels of genes instrumental in thymocyte expansion (e.g., Dll4) and positive selection (Psmb11 and Ctsl). Nevertheless, cTECs were more abundant in males than females. Accumulation of cTECs in males correlated with differential expression of genes regulating cell survival and cell differentiation. Unexpectedly, we observed that female and male sex hormones repressed promiscuous gene expression in mTECs. Since sex hormones did not affect the expression of Aire per se, they must impinge on the activity of unidentified regulator(s) of promiscuous gene expression in mTECs. The sexual dimorphism of TECs highlighted here may be mechanistically linked to the well-recognized sex differences in susceptibility to infections and autoimmune diseases. Overall design: Cortical and medullary thymic epithelial cells from 6 month-old male, female and castrated male mice were sequenced in 3 replicates (but only 2 replicates for castrated male mTECs).
Thymic Mesenchymal Cells Have a Distinct Transcriptomic Profile.
No sample metadata fields
View SamplesA soybean ortholog of the Arabidopsis MADS-domain transcription factor (called GmAGL15) enhanced somatic embryogenesis from immature cotyledon explants of soybean when expressed via the 35S promoter compared to non transgenic tissue (cultivar Jack). To better understand how this occurs an expression microarray experiment was performed.
Alterations in the transcriptome of soybean in response to enhanced somatic embryogenesis promoted by orthologs of Agamous-like15 and Agamous-like18.
Specimen part, Time
View SamplesThymocytes were extracted from a pool of three 8-12 week old C57BL-6 female mice. Cells were separated from stroma by gently crushing the thymi in between 2 microslides. RNA from thymocytes was extracted using the Trizol reagent and protocol, and analysed using the Illumina HiSeq 2000. Overall design: Transcriptomic analysis of a single replicate of thymocytes from a pool of three 8-12 week old C57BL-6 female mice, using the Illumina HiSeq 2000
Thymic Mesenchymal Cells Have a Distinct Transcriptomic Profile.
Specimen part, Cell line, Subject
View SamplesMicroarray analysis of microglia in a mouse model of amyotrophic lateral sclerosis identified the dysregulation of Brca1.
Brca1 is expressed in human microglia and is dysregulated in human and animal model of ALS.
Specimen part
View SamplesDifferences in the inherent properties of undifferentiated fat cell progenitors may contribute to the biological specificity of the abdominal subcutaneous (Sc) and visceral omental (V) fat depots. In this study, the biological characteristics of three distinct subpopulations of adipose tissue-derived stem cells (ASC), i.e. ASCSVF, ASCBottom and ASCCeiling isolated from Sc and V adipose tissue biopsies of non-obese subjects, were investigated. Genome-wide differential gene expression analysis followed by quantitative RT-PCR and analysis of cytokines in the ASC-derived conditioned medium were performed. By analysis of 28,869 annotated genes, 1,019 genes resulted differentially expressed between Sc-ASC and V-ASC. Within the Sc-ASC and V-ASC populations, 546 and 1,222, respectively, were the genes differentially expressed among ASCSVF, ASCBottom and ASCCeiling. A far more striking difference was found when the hierarchical clusters analysis was performed comparing each Sc-ASC with its own homologous V-ASC subset. mRNA levels of HoxA5, Tbx15, PI16, PITPNC1, FABP5, IL-6, IL-8, MCP-1, VEGF, MMP3, TFPI2, and ANXA10 were significantly different between Sc-ASC and V-ASC. Of the 27 cytokines measured, 14 (IL-2, IL-4, IL-5 IL-7, IL-9, IL-10, IL12, IL13, MIP1-, MIP1-, PDGF-, FGFbasic, GM-CSF, IP-10) were not released, whereas 13 were expressed (IL-1beta, IL-1ra, IL-15, IL-17, G-CSF, IFN, RANTES, TNF-, Eotaxin, IL-8, MCP-1, VEGF, IL-6), and of these, MCP-1, Eotaxin, IL-1ra, FGFbasic, IL-6, IL-8, G-CSF, and VEGF were significantly different among ASCSVF, ASCCeiling and ASCBottom of the two adipose tissue depots. These results demonstrate the existence of genetically and functionally heterogeneous fat-derived ASC populations, which may add to the complexity and specificity of Sc and V adipose tissue in humans.
Differences in gene expression and cytokine release profiles highlight the heterogeneity of distinct subsets of adipose tissue-derived stem cells in the subcutaneous and visceral adipose tissue in humans.
Specimen part
View SamplesThe identification of subtype-specific translocations has revolutionized diagnostics of sarcoma and provided new insight into oncogenesis. We used RNA-Seq to investigate samples diagnosed as small round cell tumors of bone, possibly Ewing sarcoma, but lacking the canonical EWSR1-ETS translocation. A new fusion was observed between the BCL6 co-repressor (BCOR) and the testis specific cyclin B3 (CCNB3) genes on chromosome X. RNA-Seq results were confirmed by RT-PCR and cloning the tumor-specific genomic translocation breakpoints. 24 BCOR-CCNB3-positive tumors were identified among a series of 594 sarcomas. Gene profiling experiments indicate that BCOR-CCNB3-positive cases are biologically distinct from other sarcomas, particularly Ewings sarcoma. Finally, we show that CCNB3 immunohistochemistry is a powerful diagnostic marker for this group of sarcoma and that over-expression of BCOR-CCNB3 or of a truncated CCNB3 activates S-phase in NIH3T3 cells. Thus the intrachromosomal X fusion described here represents a new subtype of bone sarcoma caused by a novel gene fusion mechanism.
A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion.
Sex, Age, Specimen part
View SamplesA mutant previously isolated from a screen of EMS-mutagenized Arabidopsis lines, per1, showed normal root hair development under control conditions but displayed an inhibited root hair elongation phenotype upon Pi deficiency. Additionally, the per1 mutant exhibited a pleiotropic phenotype under control conditions, resembling Pi-deficient plants in several aspects. Under Pi deficiency, the accumulation of Pi and iron was increased in the mutant when compared to the wild-type. Inhibition of root hair elongation upon growth on low Pi media was reverted by treatment with the Pi analog phosphite, suggesting that the mutant phenotype is not the result of a defect in Pi sensing. Reciprocal grafting experiments revealed that the mutant rootstock is sufficient to cause the phenotype. Transcriptional profiling of per1 and wild-type plants subjected to short-term Pi starvation revealed genes that may be important for the signaling of Pi deficiency. We conclude that UBP14 function is crucial for adapting root development to the prevailing local availability of phosphate.
Ubiquitin-specific protease 14 (UBP14) is involved in root responses to phosphate deficiency in Arabidopsis.
Specimen part
View Samples4 Treatment groups:
Dysregulation of gene expression in primary neuron models of Huntington's disease shows that polyglutamine-related effects on the striatal transcriptome may not be dependent on brain circuitry.
No sample metadata fields
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