|
| Status |
Public on Aug 31, 2013 |
| Title |
wrky54wrky70sid2-1_control_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
wrky54wrky70sid2-1 triple mutant, control, replicate 1
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
background cultivar: Columbia
developmental stage: Three weeks old plants
tissue: Leaves
|
| Treatment protocol |
Three weeks old plants in the soil were watered by 15% PEG6000, then collected samples at one day time point. The plants watered with water were used as control.
|
| Growth protocol |
Arabidopsis thaliana seeds were firstly put on ½ MS (Duchefa) plates and kept in +4°C for 3 or 4 days and then put the plates into in vitro room for germination. After 7 days, we transferred the green seedlings to the pots filled with mixture of peat and vermiculite (2:1) in the growth room at 22°C with 70/90% relative humidity. The light intensity is 120µmol m-2sec-1 and with a 12/12h light /dark cycle. The plants were growing for 3 or 4 weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with RNeasy® plant mini kit (Qiagen) following the manufacturer's recommendations.RNA was quantified by a NanoDrop-1000 spectrophotometer and the RNA integrity was analyzed by Agilent 2100 Bioanalyzer and the RNA was not degraded. After DNase digestion, RNA was purified by RNeasy® MinElute cleanup Kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Dye coupling reaction was performed after aRNA was synthesized, Cy3 or Hyper5 (GE Healthcare) or ARES Labeling Kit with Alexa Fluor 488 (Invitrogen) was used to label 5µg of amino allyl-modified aRNA, followed by purification of dye labelled aRNA with Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion).
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| |
|
| Hybridization protocol |
For each array, 800ng of each 3 labelled aRNA samples were mixed in 1.5ml nuclease-free microfuge tube with 10x Agilent Blocking agent, 25xAgilent Fragmentation buffer and incubated at 60°C for exactly 30 minutes. A 55µl 2xAgilent GEx Hybridization Buffer HI-RPM was added to stop the fragmentation. After pipetting to mix and centrifuging, a 100µl of the fragmentation mixture was hybridized to Agilent Arabidopsis (V4) Gene Expression Microarray for 17 hours at 65°C. After hybridiztion, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried the slide by spinning it for 1 minute in a slide centrifuge.
|
| Scan protocol |
After washing, the slides were scanned with GenePix 4200 AL scanner (Axon Instruments, Union City, CA, USA) at 635, 532 and 488 nm.
|
| Description |
Biological replicate 1 of 3, untreated,harvested after watering with water
array1.gpr
|
| Data processing |
The gpr files were produced by GenePixPro 5.0 (Axon Instruments) and imported into R 2.14 and the analysis was done with BioConductor using Limma package (http://www.springerlink.com/content/g26110k024423738/). Analyzed spots were background normalized using norm-exp model from Limma package and then different measurement groups were quantile normalized.
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| |
|
| Submission date |
Jun 06, 2012 |
| Last update date |
Aug 31, 2013 |
| Contact name |
Jing Li |
| E-mail(s) |
jing.z.li@helsinki.fi
|
| Phone |
00358919159579
|
| Organization name |
University of Helsinki
|
| Department |
Department of Bioscience
|
| Street address |
Viikinkaari 5D
|
| City |
Helsinki |
| ZIP/Postal code |
00014 |
| Country |
Finland |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE38522 |
Comparison of osmotic stress related gene expression in Col-0, sid2-1 single, wrky54wrky70 double and wrky54wrky70sid2-1 triple mutants. |
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