Dilution and amplification control study
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Stephen Fisher
PROVIDER: E-GEOD-61081 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Mouse adult female brain’s cortex (C57BL/6, Charles River Laboratories, Inc.) was isolated and stored immediately at -80°C. Subsequently, the mRNA (15µg) was isolated using TRIzol Reagent and MicroFastTrack 2.0 Kit (Invitrogen). A Sample of 5µg was assessed on Affymetrix Mouse 430.2 array. Aliquots from the leftovers of the same cortical mRNA were diluted to single-cell RNA levels (0.1, 1, and 10 pg) and independently aRNA amplified for a total of 2 and 4 rounds and assessed on Affymetrix Mouse 430.2 arrays.
2014-10-14 | GSE61081 | GEO
Project description:The HSC niche factor SCF is required for HSC maintenance. Using an Scf-GFP knockin mouse, we have identified a perivascular cell type in the bone marrow expressing high level of Scf. To characterize the novel Scf-GFP+ cells from the bone marrow, we performed microarray analysis on these cells. Total RNA were isolated from 3 independent, freshly aliquots of FACS sorted 5,000 SCF-GFP+ cells or whole bone marrow cells isolated from young adult mice. Purified RNA was amplified using the WT-OvationM-bM-^DM-" Pico RNA Amplification system (NuGEN Technologies). Sense strand cDNA was generated using WT-OvationM-bM-^DM-" Exon Module (NuGEN), then fragmented and labeled using the FL-OvationM-bM-^DM-" cDNA Biotin Module V2 (NuGEN). 2.5M-BM-5g of labeled cDNA were hybridized to Affymetrix Mouse Gene ST 1.0 microarrays.
2012-02-01 | E-GEOD-33158 | biostudies-arrayexpress
Project description:Analysis of mouse chondrocytes lacking the microRNA-140. MicroRNAs are genomically encoded small RNAs to regulate the gene expression. miR-140 shows high expression in cartilage. Results provide insight into the molecular mechanisms underlying miR-140 function in chondrocytes. Keywords: Expression profiling by array DNA microarray analysis was performed using Affymetrix mouse genome 430 2.0 array. RNA samples collected from cultured rib chondrocytes of wild-type and miR-140-/- to identify potential mRNA targets of miR-140. 2 M-BM-5g of total RNA was reverse transcribed with SuperScript II and second strand cDNA was synthesized. Biotinylated antisense cRNAs was amplified and transcribed using the GeneChip expression 3M-bM-^@M-^Y-Amplification reagent (Affymetrix). Finally, 10 M-BM-5g of cRNAs were fragmented and hybridized to GeneChip (R) Mouse Genome 430 2.0 array (Affymetrix). Microarray data were summarized by Robust Multichip Average (RMA) method and statistical analysis was performed using NIA Array Analysis (http://Igusun.grc.nia.nih.gov/ANOVA/).
2010-01-01 | E-GEOD-16007 | biostudies-arrayexpress
Project description:To assess the credibility of our experimental data in E-TABM-327 we compared transcriptional profiiles of media shifted Aspergillus fumigatus cultures using RNA that was subjected to 1, 2 or no rounds of amplification. The protocol dependent changes in log ratios were assessed
2008-01-10 | E-TABM-330 | biostudies-arrayexpress
Project description:A functional microarray targeting 24 genes involved in chlorinated solvent biodegradation pathways has been developed and used to monitor the gene expression in a contaminated site (site B) under ERD (enhanced reductive dechlorination) treatment. The microarray format provided by NimbleGen and used in this study is 12x135K. 4 M-BM-5g of labelled antisense mRNA from 3 groundwater samples were hybridized on the microarray. A 3-chip study was performed, each corresponding to hybridization with 4 M-BM-5g of labelled antisense mRNA retrieved from a monitoring well of a contaminated site (site B). Each probe (760nt) on the microarray was synthesized in eight replicates, and a total of 5,707 random probes was used to determine the background noise. Groundwater samples were collected from a contaminated site (site B) from three monitoring wells (P1, P2 and P3). P1: well located upstream to the contamination source. P2: well in the contamination source. P3 : well located downstream to the contamination source.
2012-04-02 | E-GEOD-28608 | biostudies-arrayexpress
Project description:This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 M-BM-5g/L EE2) on male medaka fertility, testicular histology and testicular gene expression. The findings demonstrate that a 14 day exposure to EE2 induced impaired male reproductive capacity and time- and dose-dependent alterations in testicular morphology and gene expression. The average fertilization rate/day following the exposure for control, 1.0 and 10.0 M-BM-5g/L EE2 was 91.3% (M-BM-14.4), 62.8% (M-BM-18.3) and 28.8% (M-BM-15.8), respectively. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. The morphologic changes observed were highly associated with gene expression changes observed using a medaka-specific microarray. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell cycle and proliferation, collagen production/extracellular matrix organization, hormone signaling, male reproduction and protein ubiquitination among others. Six month old male medaka were exposed to ethinyl estradiol (EE2) for a 14 day time period. Treatment exposures were completed in triplicate including DMSO (vehicle control), 1.0 M-BM-5g/L EE2, and 10.0 M-BM-5g/L EE2. Fish were sampled for gene expression on days 1, 7 and 14 of exposure. Five male fish were placed in 2-liter beaker replicates for each treatment and sampling time point.
2013-03-06 | E-GEOD-44859 | biostudies-arrayexpress
Project description:We used transcriptional profiling to monitor gene expression of whole olfactory bulbs at daily intervals from embryonic day 11 through birth, generating a compendium of gene expression encompassing the major developmental events of this tissue. OBs from CD1 mice (Charles River Laboratories, Wilmington, MA), were isolated from embryos daily between E11 and postnatal day zero (P0), for a total of nine time points. RNA was purified with TRIzol reagent (Invitrogen, Carlsbad, CA), subjected to two rounds of amplification, labeled, and hybridized to Affymetrix Mouse Genome 430.2 GeneChip microarrays (Affymetrix Inc., Santa Clara, CA, USA) using Affymetrix reagents and protocols (http://www.affymetrix.com). One microarray for each time point using RNA from the OBs of one individual embryo.
2011-01-04 | E-GEOD-26406 | biostudies-arrayexpress
Project description:Ketoprofen and diclofenac are non-steroidal anti-inflammatory drugs (NSAIDs) often used for similar indications, and both are frequently found in surface waters. Diclofenac affects organ histology and gene expression in fish at around 1 M-BM-5g/L. Here, we exposed rainbow trout to ketoprofen (1, 10 and 100 M-BM-5g/L) to investigate if this alternative causes less risk for pharmacological responses in fish. The bioconcentration factor from water to fish blood plasma was <0.05 (4 for diclofenac based on previous studies). Ketoprofen only reached up to 0.6M-bM-^@M-0 of the human therapeutic plasma concentration, thus the probability of target-related effects was estimated to be fairly low. Accordingly, a comprehensive analysis of hepatic gene expression revealed no consistent responses. In some contrast, trout exposed to undiluted, treated sewage effluents bioconcentrated ketoprofen and other NSAIDs much more efficiently, according to a meta-analysis of recent studies. Neither of the setups is however an ideal representation of the field situation. If a controlled exposure system with a single chemical in pure water is a reasonable representation of the environment, then the use of ketoprofen is likely to pose a lower risk for wild fish than diclofenac, but if bioconcentration factors from effluent-exposed fish are applied, the risks may be more similar. Ketoprofen (purity M-bM-^IM-%98%) (Sigma-Aldrich, Steinheim, Germany) was dissolved in water (500 mg/L concentrated solution), stirred vigorously and diluted stepwise to obtain stock concentrations (0.5 mg/L, 5 mg/L and 50 mg/L). Juvenile rainbow trout (Oncorhynchus mykiss) of both sexes (age: approximately six months, weight: 39.4M-BM-16.6g) were obtained from VM-CM-$nneM-CM-%ns fiskodling AB, Sweden, and kept in 500 L holding tanks with sand-filtered, aerated fresh water in a flow-through system for one week prior to the experiment. Eight experimental 48 L glass aquaria, semi-covered in black plastic bags to reduce visual stress from the outside (12:12 h light:dark photocycle), were supplied with filtered, aerated freshwater at a flow rate of 0.25 L/min. At the onset of the experiment 80 trout were randomly divided among the aquaria (ten fish in each). Peristaltic pumps (500 M-BM-5l/min) from each stock solution were used to reach target ketoprofen concentrations of 0 (control), 1 (low), 10 (intermediate) and 100 (high) M-BM-5g/L with two aquaria for each concentration. Water samples were taken from each aquarium at day 1, 7 and 14 and stored at -20M-BM-0C until analysis. To minimize biological variation due to variable food intake, the fish were not fed during the exposure period. It should be stressed that trout readily cope with food deprivation for much longer periods at these temperatures [Kullgren et al., 2010]. To ensure a stable water quality, temperature (12.9M-BM-10.1M-BM-0C), pH (7.4M-BM-10.2) and oxygen saturation (88.3%M-BM-11.3) were monitored every third day. The total organic carbon content of the supply water was 2.5 mg/L. At the experiment termination on day 14, the fish were killed by a rapid blow to the head and blood was collected from the caudal vessels by the use of heparinized syringes. The plasma was instantly separated by centrifugation at 10,000 rpm for 2 min and snap frozen in liquid nitrogen. The length and weight of the fish were measured and their sex determined by macroscopical observation of their gonads. Liver samples for gene expression analyses were collected and snap frozen in liquid nitrogen. Both liver and plasma samples were stored at M-bM-^HM-^R70M-BM-0C until analysis. All animal experiments were approved by the local animal committee in Gothenburg (permission number 216-2010). A 15k rainbow trout gene expression microarray was designed for the RT analyzer platform (febit, Heidelberg, Germany) using The Institute for Genomic Research (TIGR) Rainbow Trout Gene Index (RTGI) database version 7.0 (http://compbio.dfci.harvard.edu/tgi/). Details on the probe design strategy, but for eelpout (Zoarces viviparus), and transcript selection strategy are described elsewhere [Cuklev et al., 2011, Kristiansson et al., 2009, Cuklev et al., 2012]. When available, transcripts at GenBank (http://www.ncbi.nlm.nih.gov/nucleotide) were used. Results from similar microarrays, performed by our research group using the same platform, have shown good correlation with qPCR data [Cuklev et al., Kristiansson et al., 2009, Cuklev et al., 2012, Gunnarsson et al., 2009, Lennquist et al., 2011]. The frozen liver tissue was homogenized using Tissuelyser (Qiagen) and total hepatic RNA was isolated using QIAcube and RNeasyM-BM-. Plus Mini Kit (Qiagen). The RNA quantity was determined with spectophotometric measurements (Nanodrop 1000, NanoDrop Technologies) and the quality was assessed by Experion automated electrophoresis using RNA StdSense chip (Bio-Rad, Sundbyberg, Sweden). Biotinylated aRNA was synthesized using MessageAmpM-bM-^DM-" II-Biotin Enhanced Single Round aRNA Amplification Kit (AmbionM-BM-.). The aRNA samples (20 M-BM-5g) were vacuum dried in a vacuum centrifuge, dissolved in 10 M-BM-5l water and fragmented according to the manufacturerM-bM-^@M-^Ys protocol. The following steps described in this subchapter were all performed by febit. Oligonucleotide arrays were synthesized by photo-controlled in situ synthesis using the Geniom One system (febit). Each biochip consists of eight individually accessible micro-channels, each of which is referred to as a microarray in this manuscript. Four individuals (of both sexes) from each control aquarium and each high exposure concentration aquarium were included in the analysis, i.e. eight fish per exposure concentration. In total, two biochips were analyzed corresponding to 16 microarrays. Pre-hybridization and hybridization were performed based on a customized protocol, described in detail by Cuklev et al. [2011]. The samples were randomly allocated on the biochips. Signals were detected using the internal CCD-camera system of the RT analyzer instrument (febit) and quantified using the Geniom Wizard software. Integration times were 266 and 273 ms, determined automatically by the instrument software. All statistical calculations were done in R-2.12.2 (www.r-project.org) [R Development Core Team, 2010]. The quality of pre- and post-normalized arrays was verified with Box and MA plots. The data analysis was performed in the R-package LIMMA [Smyth, 2004]. Data were normalized using the M-bM-^@M-^XquantileM-bM-^@M-^Y method. Moderated t-statistics and adjusted p-values of differential expression were calculated using the empirical Bayes model. As we found no clear differences between the two control aquaria or between the two aquaria with the highest concentration of ketoprofen (100 M-BM-5g/L), all fish from the same treatment were treated as a single group, i.e. we compared one control group to one group of fish exposed to 100 M-BM-5g/L of ketoprofen.
2012-05-31 | E-GEOD-37628 | biostudies-arrayexpress
Project description:Whole fetal livers were collected from mouse fetuses at embryonic day 14.5 (E14.5), and single-cell suspensions were prepared by successive passage through 18-, 21 and 23-gauge needles. Fetal liver cells were maintained in Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100M-BM-5g/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO; Peprotech). After 5 days of culture, megakaryocytes were purified using a discontinuous bovine serum albumin gradient (BSA, SigmaM-bM-^@M-^SAldrich; 3%, 1.5%, and 0%). Total RNA was isolated with TriM-bM-^@M-^SReagent (MRC) following manufacturerM-bM-^@M-^Ys instructions, and its quality was assessed with NDM-bM-^@M-^S1000 Nanodrop (Peqlab) and on a 1.5% agarose gel. Gene expression was measured in primary megakaryocytes cultured from miR-142-/- fetal livers and wild type fetal livers at E14.5. Two independent experiments were performed.
2014-06-11 | E-GEOD-52141 | biostudies-arrayexpress
Project description:This study explores the synergistic effects of two model PAHs, an aryl hydrocarbon receptor (AHR) agonist (M-NM-2-naphthoflavone) and a cytochrome P4501A (CYP1A) inhibitor (M-NM-1-naphthoflavone), on gene expression in stage 31 embryos from two different population. One population (Elizabeth River population) is relatively resistant to the pollutants in its environment. A loop design (Figure 5) was used for the microarray hybridizations where each sample is hybridized to 2 arrays using both Cy3 and Cy5 labeled fluorophores (Kerr and Churchill, 2001). The loop consisted of Cy3 and Cy5 labeled embryo aRNAs from 4 biological samples and six different treatments (T1-T6: control, 1 M-NM-<g/L BNF, 50 M-NM-<g/L ANF, 100 M-NM-<g/L ANF, 1 M-NM-<g/L BNF + 50 M-NM-<g/L ANF, 1 M-NM-<g/L BNF + 100 M-NM-<g/L ANF). In total, 48 biological samples were hybridized to 24 microarrays. Each array had different combinations of biological samples, so that the most direct comparisons (i.e., 50 M-BM-5g/L ANF resistant embryo and 50 M-BM-5g/L sensitive embryo) are hybridized to the same array. The loop formed was T1SM-bM-^FM-^R T1R M-bM-^FM-^R T2SM-bM-^FM-^R T2R M-bM-^FM-^RT3SM-bM-^FM-^R T3RM-bM-^FM-^R T4SM-bM-^FM-^R T4RM-bM-^FM-^R T5SM-bM-^FM-^R T5R M-bM-^FM-^R T6SM-bM-^FM-^RT6RM-bM-^FM-^R T1SM-bM-^FM-^R T2S M-bM-^FM-^R T3S M-bM-^FM-^R T4S M-bM-^FM-^R T5S M-bM-^FM-^R T6S M-bM-^FM-^R T1S M-bM-^FM-^R T1R M-bM-^FM-^R T2R M-bM-^FM-^R T3R M-bM-^FM-^R T4R M-bM-^FM-^R T5R M-bM-^FM-^R T6R, where each arrow represents a separate hybridization (array) with the biological sample at the base of the arrow labeled with Cy3 and the biological pool at the head of the arrow labeled with Cy5. T1-6 is treatment, and S and R represent sensitive and resistant embryos.
2013-09-30 | E-GEOD-47654 | biostudies-arrayexpress