|
| Status |
Public on Feb 28, 2013 |
| Title |
Rosette-brc1-FR vs Rosette-brc1-WL-REP3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rosette brc1-WL [reference]
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: brc1 mutant
light: WL
tissue: Flowering rosettes comprising axillary buds but not rosette leaves
|
| Treatment protocol |
When the bolt was ~2cm long, plants were treated with 8 hours of white-light (control, Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 2.2 μeinstein · m-2 seg-1) or white light supplemented with far-red light (treatment, Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 146 μeinstein · m-2 seg-1). After the treatment, samples were collected in N2.
|
| Growth protocol |
Plants were grown in a growth chamber at 22ºC with a 16-h photoperiod (long days).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from dissected flowering rosettes comprising axillary buds but not rosette leaves were obtained using TRIzol (Invitrogen). The RNA fraction was cleaned using RNeasy midi columns (Qiagen) following manufacturer's protocol.
|
| 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 |
Rosette brc1-FR [test]
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: brc1 mutant
light: FR
tissue: Flowering rosettes comprising axillary buds but not rosette leaves
|
| Treatment protocol |
When the bolt was ~2cm long, plants were treated with 8 hours of white-light (control, Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 2.2 μeinstein · m-2 seg-1) or white light supplemented with far-red light (treatment, Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 146 μeinstein · m-2 seg-1). After the treatment, samples were collected in N2.
|
| Growth protocol |
Plants were grown in a growth chamber at 22ºC with a 16-h photoperiod (long days).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from dissected flowering rosettes comprising axillary buds but not rosette leaves were obtained using TRIzol (Invitrogen). The RNA fraction was cleaned using RNeasy midi columns (Qiagen) following manufacturer's protocol.
|
| 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 GenPix 5.1 software (Molecular Devices).
|
| Description |
Biological replicate 3 of 6: brc1 type plants illuminated with red and far-red light versus brc1 type plants illuminated with cool white fluorescent light
252116911151 Arabidopsis V4 (Ct-Cy3_Exp-Hyper5) Cuadrante 4.gpr
|
| 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 |
Feb 13, 2011 |
| Last update date |
Feb 28, 2013 |
| Contact name |
Pilar Cubas |
| Organization name |
CNB-CSIC
|
| Department |
Plant Molecular Genetics
|
| Street address |
Darwin 3
|
| City |
Campus de Cantoblanco |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE27273 |
Response to low R/FR light ratio in wt and brc1 mutant Arabidopsis axillary buds |
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