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| Status |
Public on Oct 01, 2011 |
| Title |
PindicaFromDeadBarleyRoots_5d_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Dead barley roots, P.indica inoculated, 5d
|
| Organism |
Serendipita indica |
| Characteristics |
tissue: autoclaved barley roots
age: 120hpi
medium: 1/10 PNM
|
| Treatment protocol |
For inoculation of barley roots with P. indica, the roots were dipped in a chlamydospore suspension (500,000/ml in 0.05 % Tween water) or mock inoculated and grown in sterile culture on a minimal medium (1/10 PNM) and under same growth chamber conditions as described in Schäfer et al (2009).
|
| Growth protocol |
P. indica (DSM 11827, DSMZ) was cultivated on complex medium agar plates or liquid medium as described before (Zuccaro et al., 2009). Barley seeds (Hordeum vulgare L. cv. Golden Promise) were surface sterilized with 3 % sodium hypochlorite, rinsed in water and pregerminated for 3 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Root and fungal material was harvested in liquid nitrogen after 24, 36, 48, 72, 120 and 168 hpi. For each time point roots from 15 to 20 living plants or 21 to 36 autoclaved plants were pooled. Total RNA was extracted with TRIzol (Invitrogen, Karlsruhe, Germany) following the manufacturer’s instructions. RNA quality was analysed with a 2100 Bioanalyzer (Agilent, Santa Clara, USA).
|
| Label |
Cy3
|
| Label protocol |
Two independent biological replicates for each treatment were labelled for microarrays analysis. RNA from the time points 36 and 48 hpi of P. indica colonizing roots were pooled together and referred to as the pre-penetration sample. Two more time points were selected for the hybridization, 72 hpi (early colonization) and 120 hpi (late colonization). Further RNA from 36, 48, 72 and 120 hpi of P. indica grown on CM or PNM were pooled together and used as controls, giving a total of 16 samples. The labelling preparation was performed according to Agilent’s One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) with Tecan HS Pro Hybridization protocol (version 6.0). For each reaction 500 ng of total RNA from each experiment was used.
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| |
|
| Hybridization protocol |
Cye-3-labeled probes were afterwards hybridised to 2x105k custom-designed Agilent microarrays according to Agilent's One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) protocol (version 5.7).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent High-Resolution Microarray Scanner (G2565CA) using one color scan setting for 2x105k array slides (Scan resolution 2um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
P.indica on dead barley roots, 5d after infection, replicate 2
|
| Data processing |
Microarray image files were analyzed using Agilent’s Feature Extraction software v. 10.5. For each spot, signal and background intensities were obtained. To allow for comparison of expression levels across experiments, the raw data were standardized by quantile normalization. To assess the quality of the slides diagnostic plots were generated. Intensities from same-nucleotide probes were averaged. In each group-comparison the log2-ratio between corresponding intensities was calculated and averaged over all probes of an EST. The Student´s t-statistic was applied to test EST averages for significant differences between groups. Probes with low reproducibility in the two experiments were discarded from further analysis. The selection of differentially expressed genes is based on a fold change of 2 and an absolute t-statistic of 1.96. Preliminary analysis of the microarrays data indicated that P. indica grown on 1/10 PNM was under conditions of severe starvation, therefore the data from this control were not further used in our study.
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| |
|
| Submission date |
Aug 08, 2011 |
| Last update date |
Oct 01, 2011 |
| Contact name |
Urs Lahrmann |
| Organization name |
Fraunhofer ITEM
|
| Department |
Project Group Personalized Tumor Therapy
|
| Street address |
Am Biopark 9
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13941 |
| Series (1) |
| GSE31266 |
Transcriptional profiling of Piriformospora indica colonizing living and dead barley roots |
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