|
| Status |
Public on Oct 31, 2012 |
| Title |
Leaf_Control_45days_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Control leaf, 45d, rep1
|
| Organism |
Helianthus annuus |
| Characteristics |
tissue: Leaf
age: 45d
stress: Control
|
| Treatment protocol |
Water Deficit: a mild water deficit was achieved by covering the soil with a 200 mm plastic mesh to avoid rainfall penetration into the soil. The mesh was installed 10 days before flowering, and reduced about 40% the volumetric humidity of the soil. Controlled irrigation allowed keeping the deficit level from treatment application up to the sample harvest day. Flower excision: the flower of several plants was cut with a fine scalpel 4 days before flowering, in order to suppress the reproductive sink. Control: Untreated plants were kept as controls.
|
| Growth protocol |
Field experiments were carried out at INTA Balcarce Experimental Station (37°45’S, 8°18’W) during the 2004/2005 crop season. Sunflower hybrid VDH 481 (Advanta Seeds) was sown at a 7.2 plants/m2 density and seeds emerged 11 days later. Diseases, weeds, and insects were adequately controlled. Soil fertility assured maximum yields under adequate water conditions. Rainfall was complemented with irrigation when necessary to avoid water deficit, except for the case of water deficit treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were immediately frozen in liquid nitrogen and conserved at -80°C until their processing. High quality total RNA was isolated from 100 mg of frozen using the Trizol® method (Invitrogen, Argentina). The genomic DNA was eliminated after treatment with Dnase I for 20 min at RT using DNAse I® (Invitrogen, Argentina). RNA concentration was measured using a Nanodrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware USA). Purity and integrity of total RNA was determined by 260/280 nm ratio and the integrity was checked by electrophoresis in 1% agarose gel and quality confirmed by RNA 6000 Nano Bioanalyzer (Agilent Technologies, Palo Alto, California USA) assay.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, 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 immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 45 days in control leaf
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
May 19, 2011 |
| Last update date |
Oct 31, 2012 |
| Contact name |
Paula Fernandez |
| E-mail(s) |
[email protected]
|
| Organization name |
INTA Castelar
|
| Street address |
N. Repetto y Los Reseros S/N
|
| City |
Castelar |
| State/province |
Buenos Aires |
| ZIP/Postal code |
1712 |
| Country |
Argentina |
| |
|
| Platform ID |
GPL13610 |
| Series (1) |
| GSE29390 |
First Development, Characterization and Experimental Validation of sunflower (Helianthus annuus L.) 44 K Agilent gene expression microarray |
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