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| Status |
Public on Nov 20, 2012 |
| Title |
Olive_cultivar Kalamon_90days of stress_120mM NaCl replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Olive cv. Kalamon roots 90days_120mM NaCl stress
|
| Organism |
Olea europaea |
| Characteristics |
cultivar: Kalamon (salt-tolerant)
tissue: root
|
| Treatment protocol |
Plants were irrigated daily with 150ml of half-strength Hoagland solution containing in addition 0 (control) and 120 mM/l NaCl for 15, 45, 90 days through salt stress experiment and with half strength Hoagland solution for 15 and 45 days through post-stress period.
|
| Growth protocol |
One year-old_self-rooted trees of Olea europaea L._cvs. Kalamon and Chondrolia Chalkidikis obtained from a commercial nursery and grown under identical conditions were transplanted in plastic bags containing a 1:1 mixture of perlite:sand. The young trees were irrigated daily with 150ml of half-strength Hoagland solution for one month and 35 of those of similar growth were selected for the salinity experiment while another 35 for the un-treated control. Totally 70 young trees per cultivar were used for the experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Four young trees were used to collect root tissue at each point of treatment. The root tissue was washed repeatedly with deionised water_sterilized with 0.5% sodium hypochloride_immediately ground with liquid nitrogen and stored at a -80°C freezer. Total RNA was extracted according to the method of Bachem et al._1998 and concentrated using the NucleoSpin® RNA Clean-up XS kit.
|
| Label |
Alexa 647
|
| Label protocol |
15 μg of intact total RNA were labeled with SuperScript™ Plus Direct cDNA Labeling System (Invitrogen) using Alexa Fluor®-labeled nucleotides.
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| |
|
| Hybridization protocol |
Fluorescent samples were combined and purified using the PureLink™ PCR purification System. The only modification was the use of 20μl Low temp Hybridization buffer as elution buffer. The probe was supplemented with 3.5 μl of 5 mg/ml Salmon sperm DNA and 2 μl of yeast tRNA. The mixture was heated at 60C for 5 minutes and was kept at 45C before hybridization.
The probes were placed onto the array; the slides in a sealed hybridization cassette and submerged in a 43oC water bath for 18 hours. The slides were then placed in washing solution 1 (1X SSC_0.03% SDS) and then transferred to washing solution 2 (0.2X SSC) for 5 min with agitation followed by washing solution 3 (0.05X SSC).
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| Scan protocol |
Microarrays were scanned with an Axon microarray scanner (model GenePix 4000B).
|
| Description |
KA_t_90s rep2
|
| Data processing |
The scanned images were analyzed using GenePix Pro 6.0 software. Spots were flagged as 'not found', 'absent' or 'bad'. The default local background subtraction method was used. Feature data were extracted from the raw images as GenePix Results files (.gpr) and converted to TIGR MultiExperiment Viewer (.mev) files using the ExpressConverter V2.1 tool implemented in the TM4 microarray software suite. The Integrated Intensity Value (IIV) for each spot was subsequently used as input for the normalization pipeline in MIDAS V2.21 software which included the following steps (1) locally weighted scatterplot smoothing regression (LOWESS) normalization in block mode (2) in-slide replicate analysis and (3) low-intensity filtering that removes intensity values <10000. Low-quality array elements were eliminated by applying background checking for both channels with a signal/noise threshold of 2.0 and usage of flags for both channels. One bad tolerance policy parameter was set to generous.
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| |
|
| Submission date |
Mar 01, 2012 |
| Last update date |
Nov 20, 2012 |
| Contact name |
Maria E. Manioudaki |
| E-mail(s) |
[email protected]
|
| Organization name |
MAICH
|
| Department |
Horticultural Genetics and Biotechnology
|
| Street address |
Makedonias 1
|
| City |
Chania |
| ZIP/Postal code |
73100 |
| Country |
Greece |
| |
|
| Platform ID |
GPL15288 |
| Series (2) |
| GSE36196 |
Olive (Olea europaea) cv.Kalamon: Control vs Salt-treated |
| GSE36198 |
Comparative transcriptome analysis of two olive (Olea europaea L.) cultivars in response to NaCl stress |
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