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| Status |
Public on Apr 23, 2013 |
| Title |
Genotype 9C11 Time 3 Biorep A |
| Sample type |
RNA |
| |
|
| Source name |
9C11_3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: 9C11
days_after_senescence: 31
|
| Treatment protocol |
Leaf 7 was tagged with cotton 18 days after sowing (DAS) and harvested from five randomly selected plants, 8 h into the light period, at 23, 29, 31, 33 and 35 DAS (full senescence).
|
| Growth protocol |
Arabidopsis plants were grown under a 16:8 hr light:dark cycle (lights on 04:00 to 20:00) at 20°C, 60% humidity and light intensity of 100 μmol photons.m-2.s-1. Arabidopsis seed was stratified for three days in 0.1% agar at 4°C before sowing onto Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1:1). The anac055 line (genotype IM4, col0 background) was T-DNA insertion line Salk_011069 (obtained from the Nottingham Arabidopsis Seed Centre). The anac019 dSpm insertion mutant (genotype 9C11, col5 background) was identified in a pool of SLAT line DNA screened with gene specific primers (Tissier et al., 1999).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from four individual leaves from each sampled time point (arbitrarily labelled as biological replicates A, B, C and D) using TRIzol reagent (Invitrogen), purified with RNeasy columns (Qiagen) and amplified using the MessageAmp II aRNA Amplification kit (Ambion) in accordance with the kit protocol with a single round of amplification.
|
| Label |
Cy5, Cy3
|
| Label protocol |
Cy3 and Cy5 labelled cDNA probes were prepared by reverse transcribing 5µg of aRNA with Cy3- or Cy5- dCTP (GE Healthcare) and a modified dNTP mix (10mM each dATP, dGTP and dTTP; 2mM dCTP) using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with the inclusion of RNase inhibitor (RNaseOUT, Invitrogen) and DTT. Labelled probes were purified using QiaQuick PCR Purification columns (Qiagen), freeze-dried, and resuspended in 50µl hybridization buffer (25% formamide, 5xSSC, 0.1% SDS, 0.5µg/µl yeast tRNA [Invitrogen]).
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| |
|
| Hybridization protocol |
The microarray experiments were carried out using the CATMA (version 4) microarray (Allemeersh et al., 2005; http://www.catma.org). Labelled samples were hybridized to slides overnight at 42oC.
|
| Scan protocol |
Following hybridization, slides were washed and scanned using an Affymetrix 428 array scanner at 532nm (Cy3) and 635nm (Cy5).
|
| Description |
Randomised loop design providing dye-swaps between genotypes, time points and bioreps
|
| Data processing |
Scanned data were quantified using Imagene 7.5.0 software (BioDiscovery, Inc.).
Quantified microarray data were Lowess normalied within pintip groups and within arrays, then variation due to arrays and dyes was removed by a random effects model, and averaged, in log space, using R/MAANOVA (MicroArray ANalysis Of VAriance) (Wu et al. 2003).
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| |
|
| Submission date |
Mar 14, 2013 |
| Last update date |
Apr 24, 2013 |
| Contact name |
Jonathan David Moore |
| E-mail(s) |
jonathan.moore@tgac.ac.uk
|
| Organization name |
The Genome Analysis Centre
|
| Department |
Platforms and Pipelines
|
| Street address |
Norwich Research Park
|
| City |
Norwich |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16132 |
| Series (2) |
| GSE45183 |
A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (expression between lines at timepoints). |
| GSE46318 |
A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves |
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