Arabidopsis thaliana gene expression in response to IAA challange
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE1110 | GEO | 2004/03/09
SECONDARY ACCESSION(S): PRJNA87215
REPOSITORIES: GEO
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Project description:Arabidopsis seedlings (Col-0) were grown in suspension in half-strength MS medium with agitation at ~100 rpm at ~22 C under ~50 microeinsteins m-2s-1 cool white fluorescent continuous illumination as described by (Xiao et al., Plant Physiol. 2002 Dec;130(4):2118-28). Seedlings were treated at 10-12 days by addition of freshly made IAA (0.1 or 1.0uM) to each flask, and harvested after a 1 or 3 hour incubation. Controls were not treated and harvested at 0hr. All tissue harvested. Total RNA was extracted using TRIzol (Invitrogen) as described by the manufacturer and then filtered using QIAGEN RNeasy columns. cDNA was synthesized from total RNA using a Superscript double-stranded cDNA synthesis kit (Invitrogen) and a T7-dT24 primer. cRNA was synthesized using the Enzo BioArray HighYield RNA Transcript Labeling kit (Affymetrix p/n 900182) and fragmented by Mg2+ hydrolysis. 15ug per array was hybridized overnight at 45 C. Arrays were then washed and scanned using the GeneChip FS400 fluidics station and Agilent GeneArray scanner. Images were analyzed using Affymetrix Microarray Suite 5.0, scaling to a target average intensity of 500. Spiking controls were added to the total RNA before cDNA synthesis and additional spiking samples were added to the resulting cDNA prior to cRNA synthesis. Probe samples were recovered for use in replicate hybridizations, 2 per array for a total of 4 hybridizations. The purpose of this was the statistical comparison of the two different versions of Affymetrix Arabidopsis probe arrays, AG and ATH1, as part of the process of characterization of the newer array, ATH1. A complete dose response assay using ATH1 and the data represented below may be found in series file GSE1110. In comparison table below: SIGNAL_LOG_RATIO = Mean of log to base two of the experimental divided by control signal ratios across all probe pairs in a set. CHANGE = Qualitative measurement indicating whether the probe set signal is increased (I), marginally increased (MI), not changed (NC), marginally decreased (MD), or decreased (D) as compared to a control hybridization across all probe pairs, based on a p-value calculation. change_p-value = Measures the probability that all probe pairs in the set indicate a change, with 0 indicating strong likelyhood for increase, 0.5 indicating little probability for difference, and 1 indicating strong probability for decrease. Keywords = IAA Keywords = whole plant Keywords = indole-3-acetic acid Keywords = Arabidopsis Keywords: parallel sample
2004-03-09 | GSE1111 | GEO
Project description:Arabidopsis seedlings (Col-0) were grown in suspension in half-strength MS medium with agitation at ~100 rpm at ~22 C under ~50 microeinsteins m-2s-1 cool white fluorescent continuous illumination as described by (Xiao et al., Plant Physiol. 2002 Dec;130(4):2118-28). Seedlings were treated at 10-12 days by addition of freshly made IAA (0.1 or 1.0uM) to each flask, and harvested after a 1 or 3 hour incubation. Controls were not treated and harvested at 0hr. All tissue harvested. Total RNA was extracted using TRIzol (Invitrogen) as described by the manufacturer and then filtered using QIAGEN RNeasy columns. cDNA was synthesized from total RNA using a Superscript double-stranded cDNA synthesis kit (Invitrogen) and a T7-dT24 primer. cRNA was synthesized using the Enzo BioArray HighYield RNA Transcript Labeling kit (Affymetrix p/n 900182) and fragmented by Mg2+ hydrolysis. 15ug per ATH1 array was hybridized overnight at 45 C. Arrays were then washed and scanned using the GeneChip FS400 fluidics station and Agilent GeneArray scanner. Images were analyzed using Affymetrix Microarray Suite 5.0, scaling to a target average intensity of 500. Spiking controls were added to the total RNA before cDNA synthesis and additional spiking samples were added to the resulting cDNA prior to cRNA synthesis.,In comparison table below:,SIGNAL_LOG_RATIO = Mean of log to base two of the experimental divided by control signal ratios across all probe pairs in a set.,CHANGE = Qualitative measurement indicating whether the probe set signal is increased (I), marginally increased (MI), not changed (NC), marginally decreased (MD), or decreased (D) as compared to a control hybridization across all probe pairs, based on a p-value calculation.,change_p-value = Measures the probability that all probe pairs in the set indicate a change, with 0 indicating strong likelyhood for increase, 0.5 indicating little probability for difference, and 1 indicating strong probability for decrease.
2007-07-10 | E-GEOD-1110 | biostudies-arrayexpress
Project description:Arabidopsis seedlings (Col-0) were grown in suspension in half-strength MS medium with agitation at ~100 rpm at ~22 C under ~50 microeinsteins m-2s-1 cool white fluorescent continuous illumination as described by (Xiao et al., Plant Physiol. 2002 Dec;130(4):2118-28). Seedlings were treated at 10-12 days by addition of freshly made IAA; (0.1 or 1.0uM) to each flask, and harvested after a 1 or 3 hour incubation. Controls were not treated and harvested at 0hr. All tissue harvested. Total RNA was extracted using TRIzol (Invitrogen) as described by the manufacturer and then filtered using QIAGEN RNeasy columns. cDNA was synthesized from total RNA using a Superscript double-stranded cDNA; synthesis kit (Invitrogen) and a T7-dT24 primer. cRNA was synthesized using the Enzo BioArray HighYield RNA Transcript Labeling kit (Affymetrix p/n 900182) and fragmented by Mg2+ hydrolysis. 15ug per array was hybridized overnight at 45 C. Arrays were then; washed and scanned using the GeneChip FS400 fluidics station and Agilent GeneArray scanner. Images were analyzed using Affymetrix Microarray Suite 5.0, scaling to a target average; intensity of 500. Spiking controls were added to the total RNA before cDNA synthesis and additional spiking samples were added to the resulting cDNA prior to cRNA synthesis. Probe samples were recovered for use in replicate hybridizations, 2 per array for a total of 4 hybridizations. The purpose of this was the statistical comparison of the two different versions of Affymetrix Arabidopsis probe arrays, AG and ATH1, as part of the process of characterization of the newer array, ATH1. A complete dose response assay using ATH1 and the data represented below may be found in series file GSE1110. In comparison table below:; SIGNAL_LOG_RATIO = Mean of log to base two of the experimental divided by control signal ratios across all probe pairs in a set. CHANGE = Qualitative measurement indicating whether the probe set signal is increased (I), marginally increased (MI), not changed (NC), marginally decreased (MD), or decreased (D) as compared to a control hybridization across all probe pairs, based on a p-value calculation. change_p-value = Measures the probability that all probe pairs in the set indicate a change, with 0 indicating strong likelyhood for increase, 0.5 indicating little probability for difference, and 1 indicating strong probability for decrease.
2007-10-27 | E-GEOD-1111 | biostudies-arrayexpress
Project description:Affymetrix GeneChip PM-MM probe pair is designed with the intension of measuring non-specific binding. Though the rationale behind the design id that a PM probe is expected to have a larger value than that of the MM probe, there are many exceptions in actual data. We gave an explanation for this inconsistency based on the assumption of functional states of a gene ‘ON/OFF’. Our hypothesis on PM-MM probe pairs is that the logarithmic of PM and MM values have the same distribution when gene is in OFF state. It means that the probability of MM > PM is expected to be equal to that of MM < PM for OFF genes. The validity of the hypothesis was given by inter-platform comparisons using common targets among three different types of platforms. Keywords: Affymetrix Gene Chip; Binomial distribution; Mathematical modeling; Oligonucleotide microarray; ON/OFF genes
2007-12-31 | GSE8353 | GEO
Project description:Two independent exposures (labeled T1 and T2) to pH 5.0. Samples were harvested at 30, 60, 90 and 120 minutes and used to make cDNA. Arrays are set up such that each time point was hybridized to the individual T=0 starting culture. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: all_pairs
2005-12-15 | GSE3833 | GEO
Project description:Pooled RNA samples were prepared for each of the four mouse strainsDU6, DU6i, DUKs, and DBA by epididymal fat tissues of 42 days old 20 male per lines. Pooling was used to reduce individual variability, which is high in outbred populations. For the hybridization of the tissue specific RNAs to the GeneChips, samples were prepared according to the recommendations of the Affymetrix user guide. First strand synthesis was carried out by a T7-(dT)24 primer and SuperScript II Reverse Transcriptase (Gibco BRL, Life Technologies GmbH, Eggenstein, Germany) using 10 mg whole RNA sample. Second strand synthesis was done according to the SuperScript Choice System (Gibco BRL, Life Technologies GmbH) by E. coli DNA-Polymerase I, E. coli Ligase and RNaseH. Fragment end-polishing was performed using T4-Polymerase. An in vitro transcription reaction was used to incorporate Biotin-11-CTP and Biotin-16-UTP to the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo). The fragmented cDNA was hybridized overnight at 45°C to ensure the quality of the probe. The expression levels were calculated with the GeneChip Expression Analysis softwares Data Mining Tool (Version 1.2.) and Microarray Suite (version 4.0.1.) provided by Affymetrix. Initially, hybridization signals on every GeneChip were scaled using all probe sets to minimize differences in overall signal intensities between two arrays allowing more reliable detection of biologically relevant changes in the sample. The Affymetrix decision matrix was used for the assessment of present, marginally present, and absent transcript amounts of a gene in a tested RNA. Keywords = adiposity Keywords = selected mouse lines Keywords = diabetes Keywords = insulin Keywords = leptin Keywords: parallel sample
2004-11-09 | GSE1936 | GEO
Project description:Comparison of cDNA from RNA IPed with zzYra1 to that from RNA co-IPed with zz tag alone (control IP) Keywords: equivalent probe
2002-11-25 | GSE99 | GEO
Project description:Strain background is BMA64 with RNT1 gene deleted with the TRP marker Media is SD -Trp (synthetic dextrose minimal media missing tryptophan) Cells were grown until midlog and harvested at OD600=0.4; MAS5 (Statistical Algorithm) Scaling:All Probe Sets Target Signal 500;Normalization:All Probe Sets; Alpha1=0.04; Alpha2=0.06; Tau=0.015 Strain background is BMA64 with plasmid expressing TRP marker Media is SD -Trp (synthetic dextrose minimal media missing tryptophan) Cells were grown until midlog and harvested at OD600=0.4 ; MAS5 (Statistical Algorithm) Scaling:All Probe Sets Target Signal 500;Normalization:All Probe Sets; Alpha1=0.04; Alpha2=0.06; Tau=0.015 Keywords: repeat sample
2005-07-19 | GSE2683 | GEO
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2006-01-11 | GSE4012 | GEO
Project description:Type of experiment: Comparison of liver samples between sham (control) and 20% TBSA (total burn surface area) burn rats collected at 1h, 4h, 8h, and 24h. Experimental factors: Burn injury and time Experiment design: All experimental liver samples collected after 1h, 4h, 8h, and 24h after burn injury were compared to a common reference (sham control treated as 0h time point) Bio source: Sprague-Drawley male rats weighing 150-200 g supplied through Charles river laboratories, MA (www.criver.com) Bio material manipulations: Rats (n=3 for each scald-burn and sham-burn time point) were individually housed in a temperature-controlled (25oC) and light-controlled room (12h light-dark cycle) and allowed to adjust to their new surroundings for at least 5 days prior to the experiment. Water and rat chow were provided ad libitum to the animals. On the day of the treatment, the animals were randomly divided into two groups, burned and sham-burned. The burn injury consisted of a full-skin-thickness scald burn of the dorsum, calculated to be ~ 20% of the rat’s total body surface area (TBSA), induced by immersing the designated area in boiling water for 10 s [Yamaguchi, 1997 #416]. Rats were resuscitated with an intra-peritoneal injection of sterile saline solution (1.5 mL/Kg body weight/% TBSA) immediately after burn. At each time point, three animals belonging to each group was sacrificed, the serum collected, and stored at –80oC. Hybridization extract: Total RNA was isolated from approximately 50 mg of pooled liver tissue using the Nucleospin II RNA isolation kit from Clontech (Palo Alto, CA), as per the manufacturer’s instructions. Labeling protocol: Labeling was performed as instructed by Affymetrix, Inc (www.affymetrix.com). In brief, 10 μg of total RNA was used for the double-stranded cDNA synthesis using the T7-oligo (dT) promoter primer kit (Affymetrix Inc.) and superscript RT II enzyme (Invitrogen). 1 μg of cDNA was labeled through invitro transcription reaction using T7 DNA polymerase in the presence of biotin labeled ribonucleotides. The cRNA was cleaned up using the Qiagen RNeasy columns following manufactures instructions. The cRNA (20 μg) was fragmented using the fragmentation buffer supplied as part of the Gene Chip Sample Cleanup Module (Affymetrix). Image analysis and raw data description: The images were analyzed using MAS V.0 software provided by Affymetrix, Inc. The raw data output consists of several metrices: Signal, Detection, Detection p-value, Stat pairs, Stat pairs used. Signal is a measure of the abundance of the transcript. Detection is call that indicates whether the transcript is detected (P, present), undetected (A, absent), or at the limit of detection (M, marginal). Detection p-value is a measure of the significance of the detection call. Stat pairs, is the number of probe pairs for a particular probe set on an array. Stat pairs used, is the number of probe pairs per probe set used in the analysis. Normalized and summarized data: All chips were scaled to a target intensity of 500 to account for differences between the replicate chips and their hybridization efficiencies using Affymetrix GENECHIP MAS V.0 software. The entire dataset is available at gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE802. Three filters were serially used to obtain the list of annotated genes that demonstrated differential expression in intensity between sham and burn injured animals. In the first filter, only genes that were flagged as ‘present’ by the MAS V.0 analysis software in at least one of the time points in all replicate chips were considered for further analysis. This step eliminated all genes for which the expression data was not reproducible between the replicate chips. The genes that passed this criterion were subjected to a second filter where ANOVA was performed to test each gene independently for a statistical difference in expression between groups (0, 1, 4, 8 and 24 h). The output of the analysis is the probability (p value) that a difference in expression can be observed by chance i.e., probability of getting a false positive. While the occurrence of false positives can be controlled by choosing a higher significance level (0.01 or 0.001) it also concomitantly increases the false negatives. Therefore, in this study we have chosen to work with ANOVA p-value cut-off 0.05. To reduce the occurrence of the false positives, we have used the false discovery rate (FDR) method (33) which adjusts the ANOVA p-value (cut-off 0.05) while taking the dependencies between the genes into consideration. The 695 genes that demonstrated a significant ANOVA p-value were then selected and their expression values averaged. The third filter selected 339 genes that were either up-regulated or down-regulated by at least 2-fold between any one time point (1, 4, 8 or 24h) and the 0h time point (Sham control). To determine the temporal profiles in the data, gene expression values were hierarchically clustered using the dChip software (www.dChip.org). The expression values for a gene across all samples were standardized by setting the mean to 0 and standard deviation to 1. The software then builds a hierarchical cluster tree based on centroid-linkage method using Pearson correlation coefficient as the distance metric. A set of genes were assigned to cluster groups empirically based on visual inspection of their temporal expression patterns. Keywords = Rat Keywords = burn Keywords = microarrays Keywords = fatty acids Keywords = cholesterol Keywords = inflammation Keywords: time-course
2004-05-07 | GSE802 | GEO