|
| Status |
Public on Oct 29, 2011 |
| Title |
2mM-P roots biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Medicago truncatula roots grown for 28 days at 2 mM phosphate
|
| Organism |
Medicago truncatula |
| Characteristics |
tissue: Root
genotype: Jemalong A17
age: 28 days
|
| Treatment protocol |
Plants were mycorrhized with AM fungi under conditions of phosphate limitation (20 µM phosphate). In addition, nonmycorrhizal roots grown at 20 µM phosphate as well as nonmycorrhizal roots grown at 2 mM phosphate were generated. Two different AM fungal inocula were used: Glomus mosseae granular AMF inoculum BEG 12 (Biorize R&D, Dijon, France), and Glomus intraradices inoculum (Premier Tech Biotechnologies, Rivière-de-Loup, Québec, Canada).
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| Growth protocol |
Medicago truncatula Gaertn cv Jemalong genotype A17 seeds were surface-sterilized and scarified. Plants were grown in the climate chamber (humidity: 70 %; photosynthetic photon flux: 150 μmol m-2 s-1) at a 16 h light (23 °C) and 8 h dark (18 °C) regime.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Whole-root samples were taken from frozen stocks, pooled, and ground using lysing matrix D tubes (MP Biomedicals, Illkirch, France) in a FastPrep (MP Biomedicals) prior to RNA extractions. Total RNA isolation and DNase I on-column digestion was performed via RNeasy kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA preparations were quality-checked both via spectrophotometry (NanoDrop ND-1000, Peqlab, Erlangen, Germany) and via capillary electrophoresis in RNA Nano chips (Agilent Bioanalyzer, Agilent, Böblingen, Germany), 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 added to a 250-μl hybridization cocktail also containing hybridization controls. 200 μl of the mixture was hybridized on GeneChips for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0001; GeneChip HWS kit; Affymetrix, Santa Clara, CA, USA) 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 |
Gene expression data from Medicago truncatula roots grown for 28 days at 2 mM phosphate
|
| Data processing |
Cel files obtained from Medicago GeneChip hybridizations were analysed using the Robin software (http://mapman.gabipd.org/web/guest/robin). Normalization was performed across all GeneChips using the Robust Multichip Average (RMA) algorithm. Intensity values calculated for each probe set were log2-transformed and averaged across all three biological replicates. Log2 differences between the conditions studied were evaluated statistically by applying a false discovery rate (FDR) correction for p-values implemented in Robin.
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| |
|
| Submission date |
Sep 19, 2011 |
| Last update date |
Nov 10, 2011 |
| Contact name |
Helge Küster |
| E-mail(s) |
helge.kuester@genetik.uni-hannover.de
|
| Organization name |
Leibniz Universität Hannover
|
| Department |
Institut für Pflanzengenetik
|
| Lab |
Abt. IV - Pflanzengenomforschung
|
| Street address |
Herrenhäuser Str. 2
|
| City |
Hannover |
| ZIP/Postal code |
30419 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4652 |
| Series (1) |
| GSE32208 |
Gene expression data from arbuscular mycorrhizal and phosphate-treated Medicago truncatula roots. |
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