|
| Status |
Public on Apr 05, 2010 |
| Title |
RNAi NINJA vs Wild Type (Col-0) treated with coronatine Rep 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Arabidopsis thaliana Col-0
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: Col-0 (Wild type)
|
| Treatment protocol |
The seedlins were treated and untreated with 0.5uM Coronatine during 6 hours
|
| Growth protocol |
Homozygous T3 plants silencing NINJA (RNAi-NINJA) and Col-0 sterile seeds were vernalized for 3 days at 4ºC, and grown in Petri dishes (120 mm x 120 mm), containing 1x Murashige-Skoog (MS) pH 6.0; 1 sucrose (w/v);0.7% w/v agar medium. Chamber growth conditions were 16 hours light, 8 hours dark, 22ºC, 70% relative humidity, and 250uE fluorescent illumination.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The frozen plant tissue (~100 mg) was ground in tiny mortars dipped in liquid nitrogen. The frozen powder was carefully transfered into 1.5 ml microcentrifuge tubes using small funnels and rubber spatulas also cooled with liquid nitrogen. Total RNA was extracted using TRIzol reagent (Invitrogen, 15596-026) according to manufacturer's instructions and purified with an RNeasy mini kit (Qiagen, 74104).1 ug of total RNA was amplified via the aRNA MessageAmp II kit (Ambion, 1751)
|
| Label |
Cy3
|
| Label protocol |
5 ug of aminoallyl-labeled aRNA were resuspended in 0.1 M Na2CO3 (pH 9.0) and labeled with Hyper5 and Cy3 Mono NHS Ester (CyTMDye Post-labeling Reactive Dye Pack, Amersham). Samples were purified following the manufacturer's instructions for Megaclear TM (Ambion) and Hyper5 and Cy3 incorporation was measured using 1 ul of the probe in the Nanodrop
|
| |
|
| Channel 2 |
| Source name |
Arabidopsis thaliana RNAi NINJA (At4g28910)
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
rnai: NINJA (At4g28910)
genotype: Col-0 (Wild type)
|
| Treatment protocol |
The seedlins were treated and untreated with 0.5uM Coronatine during 6 hours
|
| Growth protocol |
Homozygous T3 plants silencing NINJA (RNAi-NINJA) and Col-0 sterile seeds were vernalized for 3 days at 4ºC, and grown in Petri dishes (120 mm x 120 mm), containing 1x Murashige-Skoog (MS) pH 6.0; 1 sucrose (w/v);0.7% w/v agar medium. Chamber growth conditions were 16 hours light, 8 hours dark, 22ºC, 70% relative humidity, and 250uE fluorescent illumination.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The frozen plant tissue (~100 mg) was ground in tiny mortars dipped in liquid nitrogen. The frozen powder was carefully transfered into 1.5 ml microcentrifuge tubes using small funnels and rubber spatulas also cooled with liquid nitrogen. Total RNA was extracted using TRIzol reagent (Invitrogen, 15596-026) according to manufacturer's instructions and purified with an RNeasy mini kit (Qiagen, 74104).1 ug of total RNA was amplified via the aRNA MessageAmp II kit (Ambion, 1751)
|
| Label |
HyPer5
|
| Label protocol |
5 ug of aminoallyl-labeled aRNA were resuspended in 0.1 M Na2CO3 (pH 9.0) and labeled with Hyper5 and Cy3 Mono NHS Ester (CyTMDye Post-labeling Reactive Dye Pack, Amersham). Samples were purified following the manufacturer's instructions for Megaclear TM (Ambion) and Hyper5 and Cy3 incorporation was measured using 1 ul of the probe in the Nanodrop
|
| |
|
| |
| Hybridization protocol |
The hybridization experiment was performed according to the manufacture's protocol (Agilent technologies, Agilent 60-mer oligo microarray processing protocol: Two color microarray based gene expression analysis, G4140-90050 ver 5.7)
|
| Scan protocol |
Images from Cy3 and Hyper5 channels were equilibrated and captured with a GenePix 4000B (Axon) and spots were converted into numerical data using GenPix software (Axon).
|
| Description |
Biological replicate 2 of 4. Gene expression of RNAi NINJA (At4g28910) vs Wild Type strain (Col-0) treated 6 hours with coronatine 0.5 uM
|
| Data processing |
Raw intensities were background-substracted by NORMEXP method with a offset of 50. Signals (in log2 scale) were then normalized by LOWESS algorithm (intra-arrays normalization) followed by adjustment of their quantiles (inter-arrays normalization).
|
| |
|
| Submission date |
Dec 14, 2009 |
| Last update date |
Apr 05, 2010 |
| Contact name |
Gloria Garcia Casado |
| E-mail(s) |
ggarcia@cnb.csic.es
|
| Phone |
0034-91-5855445
|
| Organization name |
CNB-CSIC
|
| Department |
Genomica
|
| Lab |
Genomcia
|
| Street address |
C/Darwin 3
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE19455 |
NINJA connects JAZ proteins to the co-repressor TOPLESS |
|
