|
| Status |
Public on May 09, 2012 |
| Title |
PPT 24h nodules biological rep 1 |
| Sample type |
RNA |
| |
|
| Source name |
21 dpi nodules harvested from Medicago truncatula plants treated with PPT during 24 hours
|
| Organism |
Medicago truncatula |
| Characteristics |
tissue: Root nodule
genotype: Jemalong A17
age: 21 days
agent: PPT
time: 24 hrs
|
| Treatment protocol |
Nodulated plants (20 days post inoculation) were treated with a volume of the herbicide BASTA® corresponding to 0.25 mM of PPT. The herbicide was added to the nutrient solution of the aeroponic container. Root nodules were harvested just before the PPT treatment (control; PPT 0h), and at 4, 8 and 24 hours after PPT application (PPT 4h, PPT 8h, and PPT 24h) The root nodule were separated from roots at 4 ºC, pooled and immediately frozen in liquid nitrogen and stored at -80ºC.
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| Growth protocol |
Medicago truncatula Gaertn cv Jemalong genotype A17 seeds were surface-sterilized and scarified. Plants were grown aeroponically in a climate chamber (photosynthetic photon flux: 150 to 200 μmol m-2 s-1) at a 16 h light (22 °C) and 8 h dark (19 °C) regime. One month-old plants were inoculated with Sinorhizobium meliloti strain Rm1021 pXLGD4 RCR 2011(GMI51) under nitrogen limiting conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Root nodule samples were ground using a chilled mortal and pestle prior to RNA extractions. Total RNA extraction was performed via RNeasy kits (Qiagen) according to the manufacturer’s instructions. RNA preparations were quality-checked both via spectrophotometry (NanoDrop ND-1000) and via capillary electrophoresis in RNA Nano chips (Agilent Bioanalyzer), as recommended by the manufacturers.
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip Medicago Genome Arrays, according to the manufacturer’s GeneChip 3’ IVT Express kit user manual. Briefly, 100 ng of total RNA with by a RIN number (Agilent 2100 Bioanalyzer, Agilent, Böblingen, Germany) of at least 8.5 containing spiked-in poly-A+ RNA controls was used in a reverse transcription reaction (GeneChip 3’ IVT Express Kit; Affymetrix) to generate first-strand cDNA. After secondstrand synthesis, double-stranded cDNA was used in a 16 h in vitro transcription (IVT) reaction to generate aRNA (GeneChip 3’ IVT Express Kit; Affymetrix). Size distribution of in vitro transcribed aRNA and fragmented aRNA, respectively, was assessed via an Agilent 2100 Bioanalyzer, using an RNA 6000 Nano Assay.
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| |
|
| Hybridization protocol |
12.5 µg of fragmented aRNA was used in a 250-µl hybridization cocktail containing added hybridization controls. 200 µl of mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0001; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450
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| Scan protocol |
GeneChips were scanned on an Affymetrix GeneChip scanner 3000 7G.
|
| Description |
PPT 24.1
Mt_MtGeneChip0103_A012_jbecker_IGC_PP.CEL
Gene expression data from 20 dpi nodules 24 hours after PPT treatment
|
| Data processing |
CEL files of the 12 arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method available in DNA-Chip Analyzer 2010 (http://www.dchip.org, Cheng Li Lab, Harvard). Normalized CEL intensities of the arrays were used to obtain model-based gene expression indices based on a PM (Perfect Match)-only model. Subsequently log2 transformed expression data for the 50963 probe sets on the array representing Medicago truncatula transcripts were imported into Partek Genomics Suite 6.5.
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| |
|
| Submission date |
Dec 05, 2011 |
| Last update date |
May 09, 2012 |
| Contact name |
Helena Carvalho |
| E-mail(s) |
[email protected]
|
| URL |
http://www.ibmc.up.pt
|
| Organization name |
Instituto de Biologia Molecular e Celular
|
| Department |
MBNA
|
| Street address |
Rua do Campo Alegre,823
|
| City |
Porto |
| ZIP/Postal code |
4150-180 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL4652 |
| Series (1) |
| GSE34155 |
Gene expression data from phosphinothricin (PPT) treated Medicago truncatula root nodules |
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