|
| Status |
Public on Jan 01, 2018 |
| Title |
TAPG:: antisense KD_Flower pedicel_ AZ_20hr_rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Flower AZ after flower removal_20hr_rep 2
|
| Organism |
Solanum lycopersicum |
| Characteristics |
tissue: Whole flower AZ
nature of the az: partially separated
time point: 20hr
|
| Treatment protocol |
The TAPG:: antisense KD transgenic plant flower bunches containing at least two to four fresh open flowers were brought to the laboratory under high-humidity conditions. Senesced flowers and young (unopened) buds were removed, and the stem ends were trimmed. The flowers were immediately sorted by removing all but two or three fresh flowers from each stem. Bunches of five flower stalks were placed in vials with DDW. They were then placed in an observation room under conditions of 20 ± 1°C, 60 ± 10% RH, and 12 h light at intensity of 14 µmol m-2 s-2, provided by cool white fluorescent tubes. Tissue samples were taken from the AZ, of less than 1 mm thickness for each time point, excised less than 0.5 mm from each side of the visible AZ fracture. The tissues were sampled at six time points (0, 4, 8, 12, 16 and 20 h), following flower removal.
|
| Growth protocol |
Flower bunches of transgenic line of TAPG:: antisense KD gene was harvested between 9 and 11 AM from transgenic greenhouse in Israel.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was done using Qiagen method [Qiagen RNeay Plant minikit (Cat. No.74904)] as per the manufacturer protocol with DNAse treatment
|
| Label |
Cy3
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). Five hundred nanograms each of the Control and test samples were incubated with reverse transcription mix at 40°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields using Nanodrop-1000 (JH Bio) and specific activity determined based on the concentration of cRNA and dye incorporation.
|
| |
|
| Hybridization protocol |
The labeled cRNA samples were hybridized on to an Solanum lycopersicum Custom 4x180K Array designed by Genotypic Technology Private Limited. (AMADID: 043310).1650ng of cy3 labeled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327)
|
| Scan protocol |
slides were then scanned using the Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D)
|
| Description |
Gene expression in flower AZ after flower removal at 20hr
|
| Data processing |
Data extraction from Images was done using Agilent Feature Extraction software.Feature extracted data was analyzed using GeneSpring GX Version 12 software from Agilent. Normalization of the data was done in GeneSpring GX using the 75th percentile shift (Percentile shift normalization is a global normalization
|
| |
|
| Submission date |
Dec 29, 2014 |
| Last update date |
Jan 01, 2018 |
| Contact name |
Srivignesh Sundaresan |
| E-mail(s) |
srivignesh@volcani.agri.gov.il
|
| Phone |
+972-3-9683367
|
| Organization name |
Agricultural Research Organisation, The volcani center, Israel
|
| Department |
Post Harvest Technology
|
| Lab |
Dr. Shimon Meir Lab
|
| Street address |
P.O.Box 6, Bet- Dagan
|
| City |
Bet-Dagan |
| State/province |
Israel |
| ZIP/Postal code |
50250 |
| Country |
Israel |
| |
|
| Platform ID |
GPL16798 |
| Series (1) |
| GSE64564 |
Transcriptome analysis using customized AZ microarray on tomato Flower Abscission zone of transgeneic line TAPG::antisense KD gene |
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