Post-hybridization washing is an essential part of microarray experiments. Both, the quality of the experimental washing protocol and the adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities. We conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of washing cycles. Particularly, three Affymetrix GeneChip HGU133plus2 arrays were hybridized and equilibrated for 16 hours in the hybridization oven. For one of the three arrays washing and staining was performed according to the manufacturers instructions. For another array the first scan was done immediately after low stringent wash and staining without intermitting stringent washing. Then, the array was stringently washed and scanned in alternating order three more times where each washing step consists of a definite number of washing cycles. The third array was low stringently washed followed by two stringent washing cycles and staining before the first scan. Subsequently it was analogously processed as array A. All three chips are repeatedly processed in a second series of alternating wash/scan-cycles which was performed using the same protocol for each chip as in the first series as described above. As in the first series the arrays were also stained a second time to compensate for any loss of bleached fluorescent dye. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. The washing function allows calibrating probe intensities for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures especially in the limit of small and large values.
Washing scaling of GeneChip microarray expression.
Cell line
View SamplesThyroid autonomy is a frequent cause of thyrotoxicosis in regions with iodine deficiency. Epidemiological data suggest that the prevalence of thyroid autonomy is not only inversely correlated with the ambient iodine supply, but that iodide may also influence the course of pre-existing thyroid autonomy with possibly different effects on thyroid growth and function.
Effect of iodine on early stage thyroid autonomy.
No sample metadata fields
View SamplesVery small embryonic-like (VSEL) cells have been described as putatively pluripotent stem cells present in murine bone marrow and human umbilical cord blood (hUCB) and as such are of high potential interest for regenerative medicine. However, there remain some questions concerning the precise identity and properties of VSEL cells, particularly those derived from hUCB. For this reason, we have carried out an extensive characterisation of purified populations of VSEL cells from a large number of UCB samples. Consistent with a previous report, we find that VSEL cells are CXCR4+, have a high density, are indeed significantly smaller than HSC and have an extremely high nuclear/cytoplasmic ratio. Their nucleoplasm is unstructured and stains strongly with Hoechst 33342. A comprehensive FACS screen for surface markers characteristic of embryonic, mesenchymal, neuronal or hematopoietic stem cells revealed negligible expression on VSEL cells. These cells failed to expand in vitro under a wide range of culture conditions known to support embryonic or adult stem cell types and a microarray analysis revealed the transcriptional profile of VSELs to be clearly distinct both from well-defined populations of pluripotent and adult stem cells and from the mature hematopoietic lineages. Finally, we detected an aneuploid karyotype in the majority of purified VSEL cells by fluorescence in situ hybridisation. These data support neither an embryonic nor an adult stem cell like phenotype, suggesting rather that hUCB VSEL are an aberrant and inactive population that is not comparable to murine VSEL.
No associated publication
Specimen part
View SamplesThe widespread use of electricity raises the question of whether or not 50 Hz (power line frequency in Europe) magnetic fields (MFs) affect organisms. We investigated the transcription of Escherichia coli K-12 MG1655 in response to extremely low-frequency (ELF) MFs. Fields generated by three signal types (sinusoidal continuous, sinusoidal intermittent, and power line intermittent; all at 50 Hz, 1 mT), were applied and gene expression was monitored at the transcript level using an Affymetrix whole-genome microarray. Bacterial cells were grown continuously in a chemostat (dilution rate D = 0.4 h-1) fed with glucose-limited minimal medium and exposed to 50 Hz MFs with a homogenous flux density of 1 mT. For all three types of MFs investigated, neither bacterial growth (determined using optical density) nor culturable counts were affected. Likewise, no statistically significant change (fold-change > 2, P 0.01) in the expression of 4,358 genes and 714 intergenic regions represented on the gene chip was detected after MF exposure for 2.5 h (1.4 generations) or 15 h (8.7 generations). Moreover, short-term exposure (8 min) to the sinusoidal continuous and power line intermittent signal neither affected bacterial growth nor showed evidence for reliable changes in transcription. In conclusion, our experiments did not indicate that the different tested MFs (50 Hz, 1 mT) affected the transcription of E. coli.
Genome-wide transcription analysis of Escherichia coli in response to extremely low-frequency magnetic fields.
Treatment
View SamplesDiscrimination of rheumatoid arthritis (RA) patients from patients with other inflammatory/degenerative joint diseases or healthy individuals purely on the basis of genes differentially expressed in high-throughput data has proven very difficult. Thus, the present study sought to achieve such discrimination by employing a novel unbiased approach using rule-based classifiers. Three multi-center genome-wide transcriptomic data sets (Affymetrix HG- U133 A/B) from a total of 79 individuals, including 20 healthy controls (control group - CG), as well as 26 osteoarthritis (OA) and 33 RA patients, were used to infer rule- based classifiers to discriminate the disease groups. The rules were ranked with respect to Kiendls statistical relevance index, and the resulting rule set was optimized by pruning. The rule sets were inferred separately from data of one of three centers and applied to the two remaining centers for validation. All rules from the optimized rule sets of all centers were used to analyze their biological relevance applying the software Pathway Studio. The optimized rule sets for the three centers contained a total of 29, 20, and 8 rules (including 10, 8, and 4 rules for RA), respectively. The mean sensitivity for the prediction of RA based on six center-to-center tests was 96% (range 90% to 100%), that for OA 86% (range 40% to 100%). The mean specificity for RA prediction was 94% (range 80% to 100%), that for OA 96% (range 83.3% to 100%). The average overall accuracy of the three different rule-based classifiers was 91% (range 80% to 100%). Unbiased analyses by Pathway Studio of the gene sets obtained by discrimination of RA from OA and CG with rule-based classifiers resulted in the identification of the pathogenetically and/or therapeutically relevant interferon-gamma and GM-CSF pathways. First-time application of rule-based classifiers for the discrimination of RA resulted in high performance, with means for all assessment parameters close to or higher than 90%. In addition, this unbiased, new approach resulted in the identification not only of pathways known to be critical to RA, but also of novel molecules such as serine/threonine kinase 10.
Identification of rheumatoid arthritis and osteoarthritis patients by transcriptome-based rule set generation.
Sex, Age
View SamplesThis experiment series addresses the role of coactivator SRC-1/NcoA-1 for the induction of interleukin-6 (IL-6) target genes in HepG2 cells. For that purpose, HepG2 human hepatocellular carcinoma cells were manipulated to stably express an shRNA that knocks down SRC-1 expression yielding the HepG2-Src1 cells. Either unmanipulated HepG2 or HepG2-Src1 cells were then treated for various periods with IL-6.
Co-activator SRC-1 is dispensable for transcriptional control by STAT3.
No sample metadata fields
View SamplesThis is an initial experiment which was performed in order to identify novel transcriptional targets of the tumor suppressor p53
p53 activates the PANK1/miRNA-107 gene leading to downregulation of CDK6 and p130 cell cycle proteins.
Specimen part, Cell line, Treatment
View SamplesBy using high-density DNA microarrays, we analyzed the gene-expression profile of liver biopsies from young and aged donors
Impairment of Host Liver Repopulation by Transplanted Hepatocytes in Aged Rats and the Release by Short-Term Growth Hormone Treatment.
Specimen part
View SamplesT-cell acute lymphoblastic leukemia (T-ALL) cells represent developmentally arrested T-cell progenitors, subsets of which aberrantly express homeobox genes of the NKL subclass, including TLX1, TLX3, NKX2-1, NKX2-5, NKX3-1 and MSX1. Here, we analyzed the transcriptional landscape of all 48 members of the NKL homeobox gene subclass in CD34+ hematopoietic stem cells (HSCs) and during lymphopoiesis, identifying activities of 9 particular genes. Four of these were expressed in HSCs (HHEX, HLX1, NKX2-3 and NKX3-1) and three in common lymphoid progenitors (HHEX, HLX1 and MSX1). Interestingly, our data indicated downregulation of NKL homeobox gene transcripts in late progenitors and mature T-cells, a phenomenon which might explain the oncogenic impact of this group of genes in T-ALL. Using MSX1-expressing T-ALL cell lines as models, we showed that HHEX activates while HLX1, NKX2-3 and NKX3-1 repress MSX1 transcription, demonstrating the mutual regulation and differential activities of these homeobox genes. Analysis of a public T-ALL expression profiling data set comprising 117 patient samples identified 20 aberrantly activated members of the NKL subclass, extending the number of known NKL homeobox oncogene candidates. While 7/20 genes were also active during hematopoiesis, the remaining 13 showed ectopic expression. Finally, comparative analyses of T-ALL patient and cell line profiling data of NKL-positive and NKL-negative samples indicated absence of common target genes but instead highlighted deregulation of apoptosis as common oncogenic effect. Taken together, we present a comprehensive survey of NKL homeobox genes in early hematopoiesis, T-cell development and T-ALL, showing that these genes generate an NKL-code for the diverse stages of lymphoid development which might be fundamental for regular differentiation.
NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia.
Cell line
View SamplesWe used microarrays to detail the global program of gene expression underlying stemness of hematopoietic cells.
NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia.
Specimen part, Disease
View Samples