|
| Status |
Public on Aug 31, 2018 |
| Title |
PIP1-1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PIP1 peptide treatment of rosette leaves (3 hours), 2 week stage
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
tissue: Rosette leaves, 2 week stage
|
| Treatment protocol |
Seedlings were treated by spraying with an aqueous peptide solution (100 nM) supplemented with 0.02% silwet L-77 (Lehle Seeds, UK). Control seedlings were sprayed with MQ water with 0.02% silwet L-77. Whole rosettes were collected 3 h after treatment, snap-frozen in liquid nitrogen, and stored at -80°C.
|
| Growth protocol |
Seeds of Col-0 ecotype were surface-sterilised and sown out on half-strength MS plates at a density of 20 seeds per Petri dish (14 cm diameter), and stratified for 3 days at 4°C. Plates were grown under a 16 h photoperiod (70 µmol m-2 s-1) at 22°C for two weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
100 mg frozen plant tissue each from four biological replicas were homogenised using TissueLyser II (Qiagen, Hilden, Germany) for 2 x 2 minutes at 25 Hz. Total RNA was extracted with the Spectrum Plant Total RNA kit (Sigma-Aldrich, Saint Louis, USA) as described by the supplier, but with lysis solution being added to the plant tissue between the two disruption cycles. An on-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen, Hilden, Germany).
|
| Label |
Cy5
|
| Label protocol |
Total RNA (~0.2 µg) was reverse transcribed, amplified and labelled using the Low Input Quick Amp Labeling Kit, two-color, (Agilent p/n 5190-2306) (Agilent Technologies, USA). cRNA from the MQ-silwet and peptide-treated plants (PIP1 or PIP2 peptide) were alternately labelled with Cy3 or Cy5.
|
| |
|
| Channel 2 |
| Source name |
Treatment of rosette leaves with MQ water and 0.02% Silwet L-77 (3 hours), 2 week stage
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
tissue: Rosette leaves, 2 week stage
|
| Treatment protocol |
Seedlings were treated by spraying with an aqueous peptide solution (100 nM) supplemented with 0.02% silwet L-77 (Lehle Seeds, UK). Control seedlings were sprayed with MQ water with 0.02% silwet L-77. Whole rosettes were collected 3 h after treatment, snap-frozen in liquid nitrogen, and stored at -80°C.
|
| Growth protocol |
Seeds of Col-0 ecotype were surface-sterilised and sown out on half-strength MS plates at a density of 20 seeds per Petri dish (14 cm diameter), and stratified for 3 days at 4°C. Plates were grown under a 16 h photoperiod (70 µmol m-2 s-1) at 22°C for two weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
100 mg frozen plant tissue each from four biological replicas were homogenised using TissueLyser II (Qiagen, Hilden, Germany) for 2 x 2 minutes at 25 Hz. Total RNA was extracted with the Spectrum Plant Total RNA kit (Sigma-Aldrich, Saint Louis, USA) as described by the supplier, but with lysis solution being added to the plant tissue between the two disruption cycles. An on-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen, Hilden, Germany).
|
| Label |
Cy3
|
| Label protocol |
Total RNA (~0.2 µg) was reverse transcribed, amplified and labelled using the Low Input Quick Amp Labeling Kit, two-color, (Agilent p/n 5190-2306) (Agilent Technologies, USA). cRNA from the MQ-silwet and peptide-treated plants (PIP1 or PIP2 peptide) were alternately labelled with Cy3 or Cy5.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed with the Gene Expression Hybridization Kit (Agilent p/n 5188-5242). 825 ng cRNA from both MQ-silwet treated plants and peptide-treated plants were used. The cRNA mixture was fragmented and hybridized on Arabidopsis (V4) Gene Expression Microarray 4x44K arrays in a rotary oven at 65°C in ~15 h. The slides were washed with Gene Expression Wash Buffer 1 (Agilent p/n 5188-5325), Gene Expression Wash Buffer 2 (Agilent p/n 5188-5326), acetonitrile (VWR International) and Stabilization and Drying Solution (Agilent Technologies) according to the manufacturer’s instructions.
|
| Scan protocol |
The slides were scanned at 5 µm resolution on an Agilent DNA microarray scanner (Agilent Technologies).
|
| Description |
Two color microarray. Biological replicas are dye-swapped between slides.
Rosette leaves treated with PIP1 peptide for 3 hours compared to rosette leaves treated with MQ water and 0.02% Silwet L-77 for 3 hours (replica 1)
|
| Data processing |
Prior to the statistical analysis spots from control spikes, landmarks and genes with low expression (absent) were filtered out. The data were analysed using the limma package (Smyth, 2004) and the R statistical data analysis program package (R 3.0.3). No background subtraction was performed and data were normalized by the global loess normalization method. The Benjamini and Hochberg’s method to control the false discovery rate was used to identify differentially regulated genes (Benjamini and Hochberg, 1995). Genes with dye bias effects were removed and genes with an adjusted p-value less than 0.05 were regarded as significantly differentially expressed. The study is MIAME compliant.
|
| |
|
| Submission date |
Mar 09, 2016 |
| Last update date |
Aug 31, 2018 |
| Contact name |
Per Winge |
| E-mail(s) |
per.winge@ntnu.no
|
| Phone |
+47 73596229
|
| Organization name |
Norwegian University of Science and Technology
|
| Department |
Department of Biology
|
| Lab |
Bones lab
|
| Street address |
Høgskoleringen 5e
|
| City |
Trondheim |
| ZIP/Postal code |
7491 |
| Country |
Norway |
| |
|
| Platform ID |
GPL12621 |
| Series (1) |
| GSE79025 |
PAMP-induced secreted peptide 1, PIP1 (At4g28460) and PIP2 (At4g37290) are negative modulators of immunity in Arabidopsis thaliana |
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