|
| Status |
Public on Feb 13, 2013 |
| Title |
M82 1 2007 low pmt |
| Sample type |
RNA |
| |
|
| Source name |
Red ripe fruit from tomato M82 variety, biological replica 1, trial 2007
|
| Organism |
Solanum lycopersicum |
| Characteristics |
line: var. M82
tissue: epicarp and mesocarp
trial: 2007
plant replica: 1
|
| Treatment protocol |
Fruits were collected from IL 7-3 and its cultivated parent when 75% were full sized and red-ripe, softening had increased and the inside of the columella was completely red. Samples were generated by pooling ripe fruit from the same plant and discarding the seeds, jelly parenchyma, columella and placenta tissues. Samples were frozen under liquid nitrogen and stored at –80°C prior to homogenization in a Waring blender and processing for the extraction of ascorbic acid and total RNA.
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| Growth protocol |
The introgression line 7-3 (IL7-3) and its parental genotypes (cv. M82) were cultivated over consecutive years in a greenhouse at the Department of Soil, Plant, Environmental and Animal Production Sciences at the University of Naples (Portici, Italy). Six plants from IL 7-3 and from the parental cv. M82 were transferred into 20-cm pots containing a 1:1 mixture of medium sandy soil and compost at the beginning of March. Pots were distributed randomly 15 cm apart in rows 1 separated by a 50-cm 2 channel, and were supplemented with Nitrophoska Blu Spezial 12-12-17 (+2+20) (Compu) slow release fertilizer (5 g). Plants were watered twice daily using an automated irrigation device with 4 individual drip lines. Prior to flowering, the plants were supplied every two weeks with 30-10-10 liquid fertilizer (Grow More, USA). Fruits were collected at red-ripe stage.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen, homogenized and powdered tomato fruit tissue using the CTAB (hexadecyltrimethylammonium bromide) method (Griffiths et al. J Exp Bot 50: 793–798 1999). Samples were taken from IL7-3 and M82 using three plants per genotype in the 2007 and 2008 growing trials.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
Total RNA (1μg) was used as a template to synthesize antisense RNA (aRNA) with the SuperScript™ Indirect RNA Amplification System Kit (Invitrogen) incorporating Alexa Fluor 647 Reactive Dye according to manifacturer instructions.
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| |
|
| Hybridization protocol |
Prehybridization was performed by incubating the arrays with prehybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 5x Denhardt's solution, 100 ng/ul Salmon Sperm DNA, 0.05% SDS) for 60 minutes at 45 °C. 5 ug of Labeled aRNA were fragmented by incubation with Fragmentation Solution (40mM Tris Acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) for 20' at 95 °C Hybridization was performed at 45°C for 16 hours in hybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 25% DiFormamide, 100 ng/ul Salmon Sperm DNA, 0.04% SDS). Hybridization washings were performed as following: - wash with 6X SSPET wash solution (6X SSPE, 0.05% Tween-20). - wash with 3X SSPET wash solution (3X SSPE, 0.05% Tween-20). - wash with 0.5X SSPET wash solution (0.5X SSPE, 0.05% Tween-20). - wash with PBST was solution (2X PBS, 0.1% Tween-20). - 2 washes with 2X PBS
|
| Scan protocol |
After hybridization and washing, the microarray was dipped in imaging solution, covered with LifterSlip™, and then scanned using a Perkin Elmer ScanArray 4000XL and the accompanying acquisition software (ScanArray Express Microarray Analysis System v4.0). Multiple scanning at different PMT was provided for each hybridization.
|
| Data processing |
Data extraction was carried out using CombiMatrix Microarray Imager software and a median normalization of raw medians and log2 transformation were performed using SPSS v.15.0 software. Further statistical processing was performed using the two-factor ANOVA module in the TIGR Multiple Experiment Viewer Software v4.0 (http://www.tigr.org/software/tm4/).
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| |
|
| Submission date |
Apr 25, 2012 |
| Last update date |
Feb 13, 2013 |
| Contact name |
Antonio Di Matteo |
| E-mail(s) |
adimatte@unina.it
|
| Phone |
00390812539208
|
| Fax |
00390812539486
|
| Organization name |
University of Naples Federico II
|
| Department |
DiSSPAPA
|
| Lab |
Structural and functional genomics
|
| Street address |
Via Universita' 100 - Parco Gussone - Edificio 75
|
| City |
Portici (NA) |
| ZIP/Postal code |
80055 |
| Country |
Italy |
| |
|
| Platform ID |
GPL15489 |
| Series (1) |
| GSE37568 |
Comparative gene expression analysis in fruits of a tomato introgression line performing higher level of total phenolics |
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