|
| Status |
Public on Jan 01, 2012 |
| Title |
replica1 MM2d LDC1g control vs MM2d LDC 2g |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Arabidopsis thaliana MM2D callus culture
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
sample type: MM2D (Wild type culture) 1g control
cell line: MM2d
treatment: 1g control
|
| Treatment protocol |
Callus cultures were exposed to altered gravity/magnetic-inertial forces for 200 minutes. 7 conditions were used: 1 - mg*/10.1T, 2 -0.1g*/14.7T, 3 - 1g*/16.5T, 4 - 1.9g*/14.7T, 5 - 2g*/10.1T, 6 - mg/RPM inertial forces, 7 - 2g/LDC inertial forces
|
| Growth protocol |
Callus cultures of Arabidopsis thaliana were prepared one week in advance of the 200 min exposure to altered environments in the simulators from MM2d suspension cultures as described (Menges and Murray 2006). Samples were prepared in a 40.8mm height 25mm diameter tube for the Magnet (3 identical runs performed) and in two regular 90mm diameter Petri dishes for the LDC/RPM experiments (a single run performed). For all devices and conditions, the callus cultures were prepared one week before the expected experiment start and samples were preserved after treatment by quick freezing in liquid nitrogen and dry ice storage.
|
| 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 MM2D callus culture
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
cell line: MM2d
treatment: LDC (levitation) 2g
sample type: MM2D (Wild type culture) LDC 2g
|
| Treatment protocol |
Callus cultures were exposed to altered gravity/magnetic-inertial forces for 200 minutes. 7 conditions were used: 1 - mg*/10.1T, 2 -0.1g*/14.7T, 3 - 1g*/16.5T, 4 - 1.9g*/14.7T, 5 - 2g*/10.1T, 6 - mg/RPM inertial forces, 7 - 2g/LDC inertial forces
|
| Growth protocol |
Callus cultures of Arabidopsis thaliana were prepared one week in advance of the 200 min exposure to altered environments in the simulators from MM2d suspension cultures as described (Menges and Murray 2006). Samples were prepared in a 40.8mm height 25mm diameter tube for the Magnet (3 identical runs performed) and in two regular 90mm diameter Petri dishes for the LDC/RPM experiments (a single run performed). For all devices and conditions, the callus cultures were prepared one week before the expected experiment start and samples were preserved after treatment by quick freezing in liquid nitrogen and dry ice storage.
|
| 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 |
A.thaliana MM2d callus 1g vs altered gravity/magnet forces
Biological replicate 1 of 3. Transcriptional status MM2d LDC1g control vs MM2d LDC 2g
|
| 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 |
Jun 07, 2011 |
| Last update date |
Jan 01, 2012 |
| Contact name |
Raúl Herranz |
| E-mail(s) |
r.herranz@csic.es
|
| Phone |
+34918373112
|
| Fax |
+34915360432
|
| Organization name |
Centro Investigaciones Biológicas
|
| Department |
Plant Cell Nucleolus, proliferation and microgravity
|
| Lab |
Lab.205
|
| Street address |
Ramiro de Maeztu 9
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28040 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE29787 |
An environment with strong gravitational and magnetic field alterations synergizes to promote variations in Arabidopsis thaliana callus global transcriptional state |
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