|
| Status |
Public on Jan 20, 2015 |
| Title |
leaves RK32 vs Col-0-WT REP3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Col-0_WT_leaves
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype/variation: wild type
tissue: 4-weeks-old leaves
|
| Growth protocol |
4-weeks old Arabidopsis thaliana plants were grown in a growth chamber (19-23C, 85% relative humidity, 100 mEm-2 sec-1 fluorescent illumination) on a 10-hr-light and 14-hr-dark cycle. Plants were not treated prior to the RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Leaves were excised from the plants and frozen immediately in liquid nitrogen. The frozen material was grinded using mortar and pestle and the RNA was isolated with Trizol (Invitrogen. www.invitrogen.com). RNA was quantified with a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies. www.nanodrop.com). RNA quality was assessed with a 2100 Bioanalyzer from Agilent Technologies (www.agilent.com).
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified with the MessageAmp aRNA amplification kit from Ambion following the instruction manual. To allow later labeling with Cy fluorophores, aminoallyl UTP (Ambion) was added to the mix of the T7 RNA polymerase-driven aRNA amplification reaction. The amount and quality of aRNA obtained was assessed as before. The aminoallyl-labeled aRNA (10 µg) was incubated in 1 M Na2CO3 with 8 nmol of dye monofunctional NHS ester (Cy3/Cy5) RPN 5661 (Amersham Biosciences) at room temperature in the dark for 1 h. Then, 35 µL of 0.1 M sodium acetate, pH 5.2, was added and incubated for a further 5 min in the dark. The Cy-labeled aRNA was purified with the Megaclear kit from Ambion and measured with the Nanodrop ND-100 spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
RK32 mutant_leaves
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype/variation: RK32 mutant
tissue: 4-weeks-old leaves
|
| Growth protocol |
4-weeks old Arabidopsis thaliana plants were grown in a growth chamber (19-23C, 85% relative humidity, 100 mEm-2 sec-1 fluorescent illumination) on a 10-hr-light and 14-hr-dark cycle. Plants were not treated prior to the RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Leaves were excised from the plants and frozen immediately in liquid nitrogen. The frozen material was grinded using mortar and pestle and the RNA was isolated with Trizol (Invitrogen. www.invitrogen.com). RNA was quantified with a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies. www.nanodrop.com). RNA quality was assessed with a 2100 Bioanalyzer from Agilent Technologies (www.agilent.com).
|
| Label |
Cy5
|
| Label protocol |
RNA was amplified with the MessageAmp aRNA amplification kit from Ambion following the instruction manual. To allow later labeling with Cy fluorophores, aminoallyl UTP (Ambion) was added to the mix of the T7 RNA polymerase-driven aRNA amplification reaction. The amount and quality of aRNA obtained was assessed as before. The aminoallyl-labeled aRNA (10 µg) was incubated in 1 M Na2CO3 with 8 nmol of dye monofunctional NHS ester (Cy3/Cy5) RPN 5661 (Amersham Biosciences) at room temperature in the dark for 1 h. Then, 35 µL of 0.1 M sodium acetate, pH 5.2, was added and incubated for a further 5 min in the dark. The Cy-labeled aRNA was purified with the Megaclear kit from Ambion and measured with the Nanodrop ND-100 spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of dye of each aRNA labeled with either Cy3 or Cy5, ranging from 200 to 300 pmol, were mixed with 20 ug of poly(A) and 20 µg of yeast tRNA (Sigma-Aldrich) in a volume of 9 µL. To this volume, 1 µL of RNA fragmentation buffer was added (RNA fragmentation reagents; Ambion) and after 15 min at 70°C, 1 µL of stop solution. Formamide, 20x SSC, 50x Denhardt's, and 20% SDS were added to a final concentration of 50% formamide, 6x SSC, 5x Denhardt's, and 0.5% SDS. This mix was boiled for 3 min at 95°C and then added to the prehybridized slide. Hybridization took place overnight at 37°C in a hybridization chamber. Arrays were then washed for 5 min at 37°C in 0.5x SSC and 0.1% SDS, twice for 5 min at room temperature (21°C) with 0.5x SSC and 0.1% SDS, three times with 0.5x SSC at room temperature, and 5 min with 0.1x SSC. The slides were then drained with a 2000-rpm spin for 2 min. The slides were stored in darkness until they were scanned.
|
| Scan protocol |
Images from Cy3 and Cy5 channels were equilibrated and captured with a GenePix 4000B (Molecular Devices) and spots quantified using GenePix 5.1 software (Molecular Devices).
|
| Description |
Biological replicate 3 of 3: RK32 mutant versus Wild Type
RK32 vs Col-0-WT REP3
|
| Data processing |
Local background was corrected by normexp method with an offset of 50. Background corrected intensities were transformed to log scale (base 2) and normalized by loess for each array (Smyth and Speed, 2003). Finally, to have similar intensity distribution across all arrays, loess-normalized-intensity values were scaled by adjusting their quantiles (Bolstad et al., 2003).
|
| |
|
| Submission date |
Mar 06, 2012 |
| Last update date |
Jan 20, 2015 |
| Contact name |
Juan Carlos Oliveros |
| Organization name |
CNB, CSIC
|
| Street address |
Darwin 3
|
| City |
Cantoblanco |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE36308 |
Identification of novel susceptibility factors in Arabidopsis towards necreotrophic fungal pathogens. |
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