|
| Status |
Public on Feb 01, 2009 |
| Title |
Zhong You 821 stem-12 hour Infection vs control-Replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
B. napus stem tissue, infected with Sclerotinia, 12 hours after infection
|
| Organism |
Brassica napus |
| Characteristics |
B. napus (Zhong You 821), stem tissue (innoculation site + 1 cm around innoculation site, 1mm deep), plant in full flower, grown in a growth chamber, tissue taken 12 hrs after infection with Scleotinia scleotiorum
|
| Treatment protocol |
One replicate consisted of 60 plants for five time points (6, 12, 24, 48 and 72 h) and two treatments (inoculated and mock-inoculated controls). S. sclerotiorum isolate 321 generated from field canola plants were used throughout the experiment (Kohli and Kohn, 1995). When the plants were at the full flower stage, three sites of the main stem were inoculated at every other internodes with 8 mm plugs cut from the growing margin of a colony cultured on minimal salts-glucose agar (20.0 g glucose, 2.0 g NH4NO3, 1.0 g KH2PO4, 1.0 g NaOH, 0.1 g MgSO47H2O, 0.1 g malic acid and 20.0 g agar per 1000 ml distilled water) modified from (Cruickshank, 1983). Mock infected plants were inoculated with only an agar plug. Plugs were secured to the stem with Parafilm™. Each line yielded 180 inoculation sites per biological replicate or 540 sites in total. Epidermal stem tissues extending 10 mm beyond the inoculation site and 1 mm deep were harvested at 6, 12, 24, 48, and 72 h post inoculation (hpi) with a razor blade. The tissues from one biological replicate at each time point (six individual plants comprising 18 inoculation sites) were pooled as one sample. Harvested tissues were frozen immediately in liquid nitrogen and stored at -80°C.
|
| Growth protocol |
Seeds of ZY821 were sown in Redi-Earth soil mixture (Scotts Fertilizer Company) amended with coconut fibre to improve drainage and Osmocote 15-7-12 for controlled release of fertilizer. The plants were grown in a growth chamber under 200 mmol m-2 s-1 light with a 16 h photoperiod provided by fluorescent and incandescent light. The temperature was maintained at 20°C in the light and 16°C in the dark. Two seeds were planted in each of 36 cells and seedlings were thinned to one plant per cell eleven days after planting. Four week-old seedlings were vernalized in a cold room at 5 - 8°C for 4 weeks under an 8 h photoperiod and half of the previous light intensity. After vernalization, each plant was transplanted to a six-inch pot and returned to the growth chamber. Seeds of Westar were planted directly in six-inch pots and plants were grown under the same conditions as ZY821 except for the vernalization treatment. Excess plants were grown and only those with uniform stem size and architecture were used. Plants were selected for inoculation and sampling using a randomized design with three biological replicates for each cultivar.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 1 g of ground tissue using the plant RNeasy kit (Qiagen) and stored at -80°C. RNase-Free DNase was applied during RNA isolation to remove any DNA contamination (Qiagen). The concentration of total RNA was determined using a Nanodrop spectrophotometer (Nanodrop Technologies, Montchanin, DE) and verified by agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
5 mg aRNA was coupled with either Cy3 or Cy5 dye. The coupling reaction mixture was incubated at 20°C for 30 min, quenched with 4 M hydroxylamine for 15 min and purified. Cy3/Cy5 labelled aRNA was completely dried in a vacuum, dissolved in 9 ml ddH2O and fragmented by adding 1 ml of fragmentation reagent followed by incubation at 70°C for 15 min. The reaction was stopped with 1 ml of stop solution and 47 ml of hybridization solution was added.
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| |
|
| Channel 2 |
| Source name |
B. napus stem tissue, mock-infected (no sclerotinia), 12 hours after infection
|
| Organism |
Brassica napus |
| Characteristics |
B. napus (Zhong You 821), stem tissue (mock innoculation site + 1 cm around site, 1mm deep), plant in full flower, grown in a growth chamber, tissue taken 12 hrs after mock infection
|
| Treatment protocol |
One replicate consisted of 60 plants for five time points (6, 12, 24, 48 and 72 h) and two treatments (inoculated and mock-inoculated controls). S. sclerotiorum isolate 321 generated from field canola plants were used throughout the experiment (Kohli and Kohn, 1995). When the plants were at the full flower stage, three sites of the main stem were inoculated at every other internodes with 8 mm plugs cut from the growing margin of a colony cultured on minimal salts-glucose agar (20.0 g glucose, 2.0 g NH4NO3, 1.0 g KH2PO4, 1.0 g NaOH, 0.1 g MgSO47H2O, 0.1 g malic acid and 20.0 g agar per 1000 ml distilled water) modified from (Cruickshank, 1983). Mock infected plants were inoculated with only an agar plug. Plugs were secured to the stem with Parafilm™. Each line yielded 180 inoculation sites per biological replicate or 540 sites in total. Epidermal stem tissues extending 10 mm beyond the inoculation site and 1 mm deep were harvested at 6, 12, 24, 48, and 72 h post inoculation (hpi) with a razor blade. The tissues from one biological replicate at each time point (six individual plants comprising 18 inoculation sites) were pooled as one sample. Harvested tissues were frozen immediately in liquid nitrogen and stored at -80°C.
|
| Growth protocol |
Seeds of ZY821 were sown in Redi-Earth soil mixture (Scotts Fertilizer Company) amended with coconut fibre to improve drainage and Osmocote 15-7-12 for controlled release of fertilizer. The plants were grown in a growth chamber under 200 mmol m-2 s-1 light with a 16 h photoperiod provided by fluorescent and incandescent light. The temperature was maintained at 20°C in the light and 16°C in the dark. Two seeds were planted in each of 36 cells and seedlings were thinned to one plant per cell eleven days after planting. Four week-old seedlings were vernalized in a cold room at 5 - 8°C for 4 weeks under an 8 h photoperiod and half of the previous light intensity. After vernalization, each plant was transplanted to a six-inch pot and returned to the growth chamber. Seeds of Westar were planted directly in six-inch pots and plants were grown under the same conditions as ZY821 except for the vernalization treatment. Excess plants were grown and only those with uniform stem size and architecture were used. Plants were selected for inoculation and sampling using a randomized design with three biological replicates for each cultivar.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 1 g of ground tissue using the plant RNeasy kit (Qiagen) and stored at -80°C. RNase-Free DNase was applied during RNA isolation to remove any DNA contamination (Qiagen). The concentration of total RNA was determined using a Nanodrop spectrophotometer (Nanodrop Technologies, Montchanin, DE) and verified by agarose gel electrophoresis.
|
| Label |
Cy5
|
| Label protocol |
5 mg aRNA was coupled with either Cy3 or Cy5 dye. The coupling reaction mixture was incubated at 20°C for 30 min, quenched with 4 M hydroxylamine for 15 min and purified. Cy3/Cy5 labelled aRNA was completely dried in a vacuum, dissolved in 9 ml ddH2O and fragmented by adding 1 ml of fragmentation reagent followed by incubation at 70°C for 15 min. The reaction was stopped with 1 ml of stop solution and 47 ml of hybridization solution was added.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed at 37°C for 16 h in the dark, after which the slides were washed in 2 x SSC, 0.1 % SDS at 37°C for 5 min, followed by 2 washes in 1 x SSC for 2 min each and 2 washes in 0.1 x SSC for 1 min each. Washed slides were dried by centrifugation at 1000 rpm for 1 min at 20°C.
|
| Scan protocol |
The slides were scanned using a Virtek Chip Reader (Bio-Rad Laboratories). For each scan, the signal intensity of the two channels was initially adjusted based on the control spots so that an average red/green signal ratio of approximately 1 was achieved and the spots were not saturated.
|
| Description |
Biological replicate 1 of 3
The Amino Allyl MessageAmp II aRNA amplification kit was used for cDNA synthesis and RNA amplification (Ambion) with 1 mg of total RNA following the manufacturer’s instructions. In vitro transcription to prepare amplified RNA (aRNA) was conducted at 37°C for 14 h and then purified.
|
| Data processing |
Spot intensities from scanned slides were quantified using ArrayPro 4.0 (Bio-Rad Laboratories) and the data imported into the BioArray Software Environment (BASE version 1.0). Spots with intensities lower than the background were eliminated. Gene Spring 7.3 (Agilent Technologies, Inc.) was used to select spots with intensity values > 50 in at least half of the samples (slides) for further analysis. Normalization was conducted using the per spot and per-chip Lowess normalization procedure with a smooth factor of 20. A one-way ANOVA with a parametric test and variances not assumed to be equal was performed. A multiple test correction (Benjamini and Hochberg, 1995) was applied and a False Discovery Rate (FDR) of < 0.0001 was used.
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| Submission date |
Oct 17, 2008 |
| Last update date |
Oct 17, 2008 |
| Contact name |
Dwayne Hegedus |
| E-mail(s) |
[email protected]
|
| Phone |
(306) 385-9427
|
| Organization name |
Agriculture and Agri-Food Canada
|
| Street address |
107 Science Place
|
| City |
Saskatoon |
| State/province |
SK |
| ZIP/Postal code |
S7N 0X2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL7499 |
| Series (2) |
| GSE13256 |
Zhong You 821 Sclerotinia infected vs Mock infected controls in B. napus (Zhong You 821) |
| GSE13262 |
Sclerotinia infected vs Mock infected controls in B. napus Westar and Zhong You 821 |
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