|
| Status |
Public on Nov 21, 2012 |
| Title |
Berry_treated_60DAFB_vs_untreated_60DAFB_BioRep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Whole deseeded berry, NAA-treated, at 60DAFB
|
| Organism |
Vitis vinifera |
| Characteristics |
organ: whole berry (deseeded)
|
| Treatment protocol |
One-hundred bunches from fifty homogeneous plants (two bunches per plant) were treated in planta with a synthetic auxin (naphtalenacetic acid, NAA, 200 mg/L; SIGMA-N640) at the pre-véraison stage corresponding to fifty-three days after full bloom (DAFB).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from whole berries using the perchlorate method of Davies and Robinson (1996) modified by Rizzini et al. (2009).
|
| Label |
chA: Cy3
|
| Label protocol |
Total RNA was converted into target cDNA by reverse transcription using the SuperScriptTM Indirect cDNA Labeling System (Invitrogen, USA) following the manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
Whole deseeded berry, untreated, at 60DAFB
|
| Organism |
Vitis vinifera |
| Characteristics |
organ: whole berry (deseeded)
|
| Treatment protocol |
One-hundred bunches from fifty homogeneous plants (two bunches per plant) were treated in planta with a synthetic auxin (naphtalenacetic acid, NAA, 200 mg/L; SIGMA-N640) at the pre-véraison stage corresponding to fifty-three days after full bloom (DAFB).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from whole berries using the perchlorate method of Davies and Robinson (1996) modified by Rizzini et al. (2009).
|
| Label |
chB: Cy5
|
| Label protocol |
Total RNA was converted into target cDNA by reverse transcription using the SuperScriptTM Indirect cDNA Labeling System (Invitrogen, USA) following the manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
The pre-hybridization and hybridization steps were carried out in Corning® hybridization chambers with some drops of 0.3× SSC to maintain internal humidity and salts concentration, immersed in a water bath at 48 °C. The microarray was pre-hybridized for 2 h with a solution containing 5× SSC, 0.1% SDS, 1× Denhardt's, and 100 ng/μl DNA carrier. Then the slides were washed once with 0.2× SSC solution and isopropanol, and dried by centrifuging for 2 min at 2000 rpm. Before hybridization, probes were mixed to have an equal amount of each fluorescent dye, ethanol precipitated with ammonium acetate (final 2 M) and resuspended in 37 μl of hybridization solution (5× SSC, 0.1% SDS, 25% formamide, and 40 ng/μl DNA carrier). The probe was denaturated for 30 s, distributed on the spotted area of the slide, and covered with a glass cover slip. After 48 h of hybridization the slides were washed once in 1× SSC 0.2% SDS and in 0.1× SSC 0.2% SDS for 5 min and twice in 0.2× SSC for 5 min to eliminate completely SDS residues that may cause shadows on the spotted area.
|
| Scan protocol |
The microarrays were scanned with a two-channel confocal microarray scanner (ScanArray® Lite, Perkin-Elmer) using its dedicated software (ScanArray Express 3.0.0., Perkin-Elmer). The laser power was set between 67 and 79% of maximum and the photomultiplier tube (PMT) was set between 69 and 79% of maximum. The excitation/emission settings were 543/570 nm for Cy3 and 633/670 nm for Cy5. After laser focusing and balancing of the two channels, scans were conducted at a resolution of 5 μm. For any scan, two separate 16-bit TIFF images were produced.
|
| Description |
Comparison between gene expression profiles of NAA-treated and untreated berries at 3 days after the treatment (60 DAFB) - Biological replicate 1
|
| Data processing |
Raw hybridization data were quality-filtered, background-subtracted, and intra-array normalized with the loess method. Calculations were all carried out with the package limma and other basic statistical functions of R for Mac OS X v2.13.1 (http://www.r-project.org/).
|
| |
|
| Submission date |
Apr 17, 2012 |
| Last update date |
Nov 22, 2012 |
| Contact name |
Alessandro Botton |
| E-mail(s) |
alessandro.botton@unipd.it
|
| Organization name |
University of Padova
|
| Department |
Agronomy, Food, Natural resources, Animals and Environment
|
| Street address |
Agripolis - viale dell'università 16
|
| City |
Legnaro |
| State/province |
Padova |
| ZIP/Postal code |
35020 |
| Country |
Italy |
| |
|
| Platform ID |
GPL15453 |
| Series (1) |
| GSE37341 |
Grape berry: control vs NAA-treated |
|
