Project description:Prior to generation of microarray data for a Top-down Systems analysis of influenza A infection, we evaluated the degree and origin of technical variation in gene expression microarray data from mouse lungs and compared this to inter-animal variation. Technical variation in these microarray data in the context of inter-animal variation supported a choice of biological rather than technical replicates.
Project description:Prior to generation of microarray data for a Top-down Systems analysis of influenza A infection, we evaluated the degree and origin of technical variation in gene expression microarray data from mouse lungs and compared this to inter-animal variation. Technical variation in these microarray data in the context of inter-animal variation supported a choice of biological rather than technical replicates. A) One mouse (260) was infected with 0.6LD50 of the mouse pathogenic H1N1 strain PR8. Lung tissues were collected at 72h post infection and processed to yield whole lung RNA that was used for microarray analysis. The RNA was extracted in 3 individual batches (I, II, III), each batch of RNA was amplified in 2 separate batches (1,2), and the individual batches of amplified product were again hybridized in 2 batches. This dataset contains 18 microarrays covering 1 experimental condition with 1 biological replicate and a matrix of technical replicates. B) 3 mice (245, 246, 247) were infected with 0.6LD50 of the mouse pathogenic H1N1 strain PR8. Lung tissues were collected at 72h post infection and processed to yield whole lung RNA that was used for microarray analysis. From each animal, RNA was extracted, the RNA was amplified in three separate batches (A, B, C), and the individual batches of amplified product were again hybridized in 2 batches. This dataset contains 24 microarrays covering 1 experimental condition with 3 biological replicates and a matrix of technical replicates. Datasets A) and B) are linked to two biological datasets - one comparing the transcriptomes of 5 different cell types isolated from individual lungs of influenza A-infected or control animals (contains 75 microarrays covering 25 experimental conditions) and one comprising whole lung microarrays from sham-infected or influenza H1N1-infected animals (contains 138 microarrays, 19 experimental conditions, 7 biological replicates).
Project description:Phosphorylation of proteins on serine, threonine, and tyrosine residues is a ubiquitous post-translational modification that plays a key part of essentially every cell signaling process. It is reasonable to assume that inter-individual variation in protein phosphorylation may underlie phenotypic differences, as has been observed for practically any other molecular regulatory phenotype. However, we do not know much about the extent of inter-individual variation in phosphorylation because it is quite challenging to perform a quantitative high throughput study to assess inter-individual variation in any post-translational modification. To test our ability to address this challenge with current technology, we quantified phosphorylation levels for three fully sequenced human cell lines within a nested experimental framework, and found that genetic background is the primary determinant of phosphoproteome variation. We uncovered multiple functional, biophysical, and genetic associations with germline driven phosphopeptide variation (though the small sample size in this ‘pilot’ study limits the applicability of our genetic observations). Among these associations were variants affecting protein levels or structure, with the latter presenting, on average, a stronger effect. Interestingly, we found evidence that is consistent with a phosphopeptide variability buffering effect endowed from properties enriched within longer proteins. We also undertook a thorough technical assessment of our experimental workflow to aid further efforts. Taken together, this work provides the foundation for future work to characterize inter-individual variation in post-translational modification as well as reveals novel insights into the nature of inter-individual variation in phosphorylation.
Project description:Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq proved more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.
Project description:The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. Results: We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Conclusions: Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. 140 Affymetrix Snowball microarray were analysed from 5 different breed (DR, LR, LW, PIE and HAM). 5 pig per breed were used and cells were harvested from Lungs, blood and bone-marrow (AM, MDM and BMDM). Cells were left untreated (0h) or stimulated with LPS Salmonella enterica serotype minnesota Re 595 - 100ng/ml (7h)
Project description:Purpose of the current study was to identify miRs that can differentiate between patients with and without local relapse after breast conserving therapy in early stage breast cancer, which is especially important in the context of individualized treatment selection. The study was conducted in a pilot and a validation phase. First, 32 patients were screened for the most de-regulated miRs in a panel of 1250 miRs by microarray technology. Second, the candidate miRs were analyzed in an independent cohort of 115 patients using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
Project description:In the teleost fish Fundulus heteroclitus there is extensive variation in gene expression among individuals within and between populations. Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization. In this manuscript we analyze the relationship between the amount of mRNA and the fluorescent signal from the microarray hybridizations demonstrating that for a wide-range of mRNA concentrations the fluorescent signal is a linear function of the amount of mRNA. Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression. However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid. To examine both technical and stochastic biological variation, mRNA expression was measured from the same five individuals over a six-week time course. There were few, if any, meaningful differences in gene expression among time points. Thus, microarray measures using standard laboratory procedures can be precise and quantitative and are not subject to significant random biological noise.
Project description:Knowledge of the biological and technical variation for fermentor-grown Aspergillus niger cultures is needed to design DNA microarray experiments properly. We cultured A. niger in batch-operated fermentor vessels and induced with D-xylose. Transcript profiles were followed in detail by qPCR for 8 genes. A variance components analysis was performed on these data to determine the origin and magnitude of variation within each process step for this experiment. 6 Fermentor cultures were selected to determine technical and biological variation for all 14554 ORFs present on this array type. Keywords: Validation of microarrays; variation analysis; experimental design
2008-12-31 | GSE11405 | GEO
Project description:On the relevance of technical variation due to building pools in microarray experiments