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.
No sample metadata fields
View SamplesThe S1 and S3 erythroid developmental subsets were isolated using flow cytometry and the cell surface markers CD71 and Ter119 as described by Pop et. al. 2010 (PMID: 20877475)
Global DNA demethylation during mouse erythropoiesis in vivo.
Specimen part
View SamplesGene expression microarrays have made a profound impact in biomedical research. The diversity of platforms and analytical methods has made comparison of data from multiple platforms very challenging. In this study, we describe a framework for comparisons across platforms and laboratories. We have attempted to include nearly all the available commercial and in house platforms. Using probe sequences matched at the exon level improved consistency of measurements across the different microarray platforms compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values as confirmed by QRT-PCR. Concordance of measurements was higher between laboratories on the same platform than across platforms. We demonstrate that, after stringent pre-processing, commercial arrays were more consistent than in-house arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms.
A sequence-oriented comparison of gene expression measurements across different hybridization-based technologies.
No sample metadata fields
View SamplesGene expression microarrays have made a profound impact in biomedical research. The diversity of platforms and analytical methods has made comparison of data from multiple platforms very challenging. In this study, we describe a framework for comparisons across platforms and laboratories. We have attempted to include nearly all the available commercial and in-house platforms. Using probe sequences matched at the exon level improved consistency of measurements across the different microarray platforms compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values as confirmed by QRT-PCR. Concordance of measurements was higher between laboratories on the same platform than across platforms. We demonstrate that, after stringent pre-processing, commercial arrays were more consistent than in-house arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms.
A sequence-oriented comparison of gene expression measurements across different hybridization-based technologies.
No sample metadata fields
View SamplesThe roles of RNA-binding proteins as chaperones in the lifecycles of mRNAs are not well understood. The mammalian mitochondrial genome has been compressed over evolution to a size of just 16 kb, nevertheless the expression of its genes requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, providing an opportunity to use it as a model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA-protein interactions guided by the LRPPRC/SLIRP complex in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC/SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization and polyadenylation. Our findings reveal a general mechanism where extensive RNA-protein interactions ensure that RNA is accessible for its biological functions. Overall design: RNase footprinting of LRPPRC and SLIRP knockout and control mice, in technical duplicate.
LRPPRC-mediated folding of the mitochondrial transcriptome.
Specimen part, Cell line, Subject
View SamplesChronic alcohol consumption can lead to alchohol-related brain damage (ARBD). Despite the well known acute effects of alcohol the mechanism responsible for chronic brain damage is largely unknown. Pathologically the major change is the loss of white matter while neuronal loss is mild and restricted to a few areas such as the prefrontal cortex. In order to improve our understanding of ARBD pathogenesis we used microarrays to explore the white matter transcriptome of alcoholics and controls.
Comorbidities, confounders, and the white matter transcriptome in chronic alcoholism.
Specimen part, Disease, Disease stage
View SamplesDifferential gene expression as a consequence of PTCD1 loss Overall design: We used RNA from control and PTCD1 knockout mice to investigate changes at the RNA level in response to PTCD1 loss
PTCD1 Is Required for 16S rRNA Maturation Complex Stability and Mitochondrial Ribosome Assembly.
Specimen part, Subject
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Multi-omic integrated networks connect DNA methylation and miRNA with skeletal muscle plasticity to chronic exercise in Type 2 diabetic obesity.
Sex, Specimen part, Treatment
View SamplesAnalysis of skeletal muscle gene expression from type 2 diabetic volunteers before and after 16 weeks of chronic exercise training (two groups, one undergoing aerobic ecercise and the other resistance training exercise)
Multi-omic integrated networks connect DNA methylation and miRNA with skeletal muscle plasticity to chronic exercise in Type 2 diabetic obesity.
Specimen part
View SamplesWe report that Dnmt1 is crucial during perinatal intestinal development. Loss of Dnmt1 in intervillus progenitor cells causes global hypomethylation, DNA damage, premature differentiation, and apoptosis, and consequently, loss of nascent villi. We further confirm the critical role for Dnmt1 during crypt development using the in vitro organoid culture system, and illustrate a clear differential requirement for Dnmt1 in immature versus mature organoids. These results demonstrate an essential role for Dnmt1 in maintaining genomic stability during intestinal development and the establishment of intestinal crypts. Overall design: We performed RNA-Seq of control and Dnmt1-ablated intestinal progenitor cells isolated from parrafin embedded tissues by laser capture microdissection (LCM).
Dnmt1 is essential to maintain progenitors in the perinatal intestinal epithelium.
No sample metadata fields
View Samples