|
| Status |
Public on Apr 10, 2014 |
| Title |
FLIGHT TAGES 2B plate6 HYPOCOTYLS Rep2 |
| Sample type |
RNA |
| |
|
| Source name |
solid 0.5x MS media plates; Advanced Biological Research System (ABRS); The EXPRESS Rack
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
cultivar: 60% WS, 40% Col-0
material type: plant tissue
organism part: hypocotyls
age: seeds germinated with light stimulus 12 days into the flight
biosource type: in-flight fixed sample
|
| Treatment protocol |
Experiment Unique Equipment and Environmental Conditions:Both the flight and ground control components of the experiment were grown in specialized growth chamber units developed by Kennedy Space Center referred to as ABRS (Advanced Biological Research System) growth chamber. Seeded plates were unwrapped from their light-tight coverings and installed in a specialized GFP imaging system (GIS) designed to hold six plates – one in the direct line of fluorescent excitation LEDs and the imaging camera, plus five additional plates. GFP expression data were collected on orbit form the imaging plate, and after 12 days of growth, all plates were harvested on orbit to RNAlater-filled KSC Fixation Tubes (KFTs) and then stowed below -40°C on station. The Ground Control was housed in the Orbital Environmental Simulator (OES) chamber in the Spaceflight Life Sciences Laboratory at Kennedy Space Center. The Ground Control was initiated with a precise 48 hour delay to enable the OES environment to be programed with those environmental conditions that can be replicated on the ground, such as cabin temperature and CO2 levels, taken from ISS telemetry. Sample Preparation: Plants harvested to RNAlater-filled KFTs were returned from ISS stowage with Orbiter Atlantis for the return flight of STS-132 and transferred to the PIs. Total RNA was isolated form sets of intact 12 day old seedlings, and from seedlings separated into three tissue types: leaves (cut from the plant at base of each petiole), hypocotyls (the region between the root/shoot junction and the start of the leaf rosette) and roots.
|
| Growth protocol |
Plant Lines and Seed Dormancy: Arabidopsis thaliana (Arabidopsis) plants were grown in a vertical configuration on the surface of 10 cm2 solid media plates. Three GFP (Green fluorescent Protein reporter gene lines were used: Adh::GFP (alcohol dehydrogenase promoter), DR5::GFP (synthetic auxin response element) and UB::GFP (35sCaMV constitutive promoter). Seeds, and the seeded plates, were prepared in such a way as to maintain dormancy until the initiation of the experiment on orbit. Seeds were surface sterilized with 75% ethanol for 10 minutes, dried on sterile filter paper and stored at 4°C until use. Immediately before planting onto media plates, a small amount of seed was suspended in sterile water and the seeds were dispersed individually to the surface of the plate. The plate was then immediately wrapped in light-tight black cloth (Duvatyne) and stored at room temperature until launch. After launch (Shuttle launch STS-131, Orbiter Discovery, April, 2010) and transition to the International Space Station (ISS), the wrapped plates were stowed at ambient temperature for 12 days until the initiation of the experiment on orbit. The dry-sterilized seeds remained dormant until activated by exposure to light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted using RNeasy Mini Kit (QIAGEN)
|
| Label |
biotin
|
| Label protocol |
cDNA was synthesized using Ovation Pico WTA System (NuGEN Technologies Inc.) and cDNA was labeled using Encore Biotin Module (NuGEN Technologies Inc.)
|
| |
|
| Hybridization protocol |
Amplified and labeled cDNA (5 µg/sample) was fragmented and hybridized with rotation onto Affymetrix GeneChip Arabidopsis ATH1 Genome Arrays for 16h at 45°C. Arrays were washed on a Fluidics Station 450 (Affymetrix) with the Hybridization Wash and Stain Kit (Affymetrix) and the Washing Procedure FS450_0004.
|
| Scan protocol |
scan was performed using Affymetrix GeneChip Scanner 3000 7G
|
| Data processing |
The MAS5 data was generated by using the Affymetrix Expression Console software using default analysis settings and global scaling as normalization method.
|
| |
|
| Submission date |
Apr 09, 2014 |
| Last update date |
Mar 03, 2017 |
| Contact name |
Robert J. Ferl |
| E-mail(s) |
ferllabuf@gmail.com
|
| Phone |
352-273-8030
|
| Organization name |
University of Florida
|
| Department |
Horticultural Sciences
|
| Lab |
Ferl's lab
|
| Street address |
1301 Fifield Hall PO Box 110690
|
| City |
Gainesville |
| State/province |
Florida |
| ZIP/Postal code |
32611 |
| Country |
USA |
| |
|
| Platform ID |
GPL198 |
| Series (1) |
| GSE56659 |
The Arabidopsis spaceflight transcriptome: a comparison of whole plants to discrete root, hypocotyl and shoot responses to the orbital environment |
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