|
| Status |
Public on Mar 28, 2012 |
| Title |
Myc DMI3 roots early stages biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Gigaspora margarita-colonized Medicago truncatula root fragments at 5 days post inoculation
|
| Organism |
Medicago truncatula |
| Characteristics |
tissue: Root
genotype: Jemalong DMI3
age: 5 days
treatment: Gigaspora margarita-colonized
|
| Treatment protocol |
Hairy roots were inoculated with 8 spores of G.margarita BEG 3. Fungal spores originated from pot cultures of mycorrhizal clover, vernalized at 4°C for 2 weeks and surface sterilized with 3% (w/v) chloramine and streptomycin.
|
| Growth protocol |
Hairy roots of Medicago truncatula Gaertn cv Jemalong genotypes A17 and DMI3 were maintained in Petri dishes containing M medium and covered with Biofolie 25™ (Sartorius).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Root fragments were taken from frozen stocks, and ground using using a Retsch® ball mill for 2 min. RNA was then extracted using the total RNA isolation system (Promega). Integrity of RNA samples was checked using a Biorad Experion Bioanalyzer, while RNA purity was determined by a NanoDrop, ensuring spectrophotometric ratios of A260nm/A280nm ~ 2 and A260nm/A230nm ≥ 2. Removal of genomic DNA was done via the Turbo DNA-free™ reagent (Ambion, Austin, TX, USA) following the manufacturer’s instructions. Absence of genomic DNA was verified by RT-PCR using a One-step kit (Qiagen) with specific primer for M. truncatula elongation factor alpha (MtTefalpha).
|
| 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.
|
| |
|
| 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.
|
| Scan protocol |
GeneChips were scanned on an Affymetrix GeneChip scanner 3000 7G.
|
| Description |
Gene expression data from Gigaspora margarita-colonized Medicago truncatula root fragments at 5 days post inoculation
|
| 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.
|
| |
|
| Submission date |
Dec 21, 2011 |
| Last update date |
Mar 28, 2012 |
| 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) |
| GSE34617 |
Gene expression during early stages of arbuscular mycorrhizal root formation in Medicago truncatula. |
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