|
| Status |
Public on May 15, 2013 |
| Title |
ColdShock_K326_lea_0h_morning_r1 |
| Sample type |
RNA |
| |
|
| Source name |
Leaves
|
| Organism |
Nicotiana tabacum |
| Characteristics |
exp.type: Induced Stress
stress: ColdShock
harvest time: morning
pos-harvest time: 0h
developmental stage: four leaves visible
provider: DNAVision
|
| Treatment protocol |
Leaves were quickly chilled for 10 minutes in a blast chiller at a temperature included between 0 Celsius and 5 Celsius, monitored to avoid frost. After fast chilling, the samples were incubated at the same temperature for 5 hours or 24 hours before to be frozen in liquid nitrogen. Two harvesting times, one in the morning (7:30am) and one in the afternoon (1:00pm)
|
| Growth protocol |
Tobacco seeds ofK326 varieties were germinated in floating trays and grown 6 weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 100 ug of plant tissue was placed in a 1.5 ml Eppendorf tube (1/3 in volume), and snap frozen in liquid nitrogen. RNA extraction was performed by two methods: Trizol(R) for microarray analysis and Qiagen RNeasy Plant mini kit for semi-quantitative RT-PCR. In case of RNA extraction for microarray experiment, 400 ul of Trizol(R) reagent (Invitrogen) was added to the frozen plant tissue and the sample was ground to homogeneity before an additional 600 ul of Trizol(R) was added. Samples were vortexed for 15 sec, then incubated at for 5 min room temperature. 200 ul of chloroform was added and the tubes were gently mixed before centrifugation (12,000 x g) at a temperature of 4 Celsius for 15 min. The aqueous phase was transferred to an Eppendorf tube containing 500 ul of isopropyl alcohol. After 15 min incubation on ice, RNA was pelleted by centrifugation at a temperature of 4 Celsius for 10 min. The RNA pellet was finally washed with 1 ml of 70% ethanol, and centrifuged for five minutes a 7,500 x g. After air-drying, RNA was resuspended in 50 ul of nucleotide-free H2O. RNA concentration was analyzed by measuring optical density (OD) at 260 nm. RNA quality control was determined by OD260/OD280 and using Agilent 2100 Bioanalyzer before cDNA preparation.
|
| Label |
biotin
|
| Label protocol |
Standard Affymetrix labeling procedure using biotin
|
| |
|
| Hybridization protocol |
Frozen samples (-80 Celsius) packed in dry ice were sent to DNA-Vision (Charleroi, Belgium). Total RNA isolation using a Trizol(R) method (Invitrogen 155596-018), microarray hybridization and quality checked by Agilent 2100 Bioanalyzer were executed at DNA-Vision and. Affymetrix hybridizations were performed using Affymetrix kits with catalog numbers 900652 and 900454; probe labeling was checked as suggested by the manufacturer.
|
| Scan protocol |
Standard Affymetrix scanning procedure
|
| Description |
QC Passed
|
| Data processing |
An in-house QC pipeline was developed to assess the quality of the gene-expression data. In addition to the standard quality metrics suggested by Affymetrix, an in-house QC metrics including probe-level models, Normalized Unscaled Standard Error (NUSE) and Relative Log Expression (RLE) plots, and the analysis of DABG results were used. The QC pipeline involves a combination of Affymetrix Power Tools (APT) and Bioconductor packages, for which the Tobacco Exon Array (TobArray520623F) cdf environment was created.
As the exon array design had no mismatch probes, summarization was performed using Robust Multiarray Average (RMA) method. A total of 272,472 probesets expression values were generated, and DABG P-values were computed to assess the significance of the signal obtained for each probeset. This involved the background probes that are spread over the chip. Those random probes have a varying GC content. Once the expression values were available, differential gene analysis was performed using moderated t-statistics in linear model LIMMA. In addition, gene sets were defined by first annotating probesets by homology to Arabidopsis thaliana genes and using Arabidopsis thaliana gene sets, and mean-rank gene-set enrichment analysis was performed.
|
| |
|
| Submission date |
Mar 07, 2013 |
| Last update date |
May 15, 2013 |
| Contact name |
Sam Ansari |
| E-mail(s) |
sam.ansari@pmi.com
|
| Organization name |
Philip Morris Products SA
|
| Department |
Research & Development
|
| Lab |
Biological Systems Research
|
| Street address |
Quai Jeanrenaud 5
|
| City |
Neuchatel |
| State/province |
Neuchatel |
| ZIP/Postal code |
2000 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16290 |
| Series (1) |
| GSE44938 |
ExpressionData - A public resource of high quality datasets representing gene expression across tissues, conditions, diseases and genotypes. |
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