|
| Status |
Public on Nov 29, 2023 |
| Title |
leaf, 10 hpi, resistant, rep2 |
| Sample type |
RNA |
| |
|
| Source name |
leaf, resistant, 10 hpi, rep-2
|
| Organism |
Triticum aestivum |
| Characteristics |
tissue: Leaf
age: 25 day
phenotype: resistant NIL
infection: stem rust race
time: 10 hpi
|
| Treatment protocol |
25 days old seedlings were injected with stem rust spore suspension ( ~ 6X10¬5 spores/ml in water and 1 ppm tween 20 detergent) using a hypodermal syringe, plants were then initially kept undisturbed for 24 hours in dark and 90% humidity, and then to normal growth (16h light, 8 h dark) conditions. Leaf tissue was collected at 0, 10 & 72 hpi time points.
|
| Growth protocol |
Wheat seed were sown in small pots in equal mix of autoclaved soil and soil rite. The pots were kept in climate controlled growth chamber with 16h light and 8 h dark cycle with 80-90 % humidity. Temprature was maintained at 25ºC during light phase and 20ºC during dark phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the TRIzol following the manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). The total RNA were reverse transcribed at 40°C using oligo dT primer tagged to a T7 polymerase promoter and converted to double stranded cDNA. 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 using Qiagen RNeasy columns (Qiagen, Cat No: 74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000.
|
| |
|
| Hybridization protocol |
Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of (Agilent Technologies, In situ Hybridization kit, 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 (Agilent Technologies, Part Number 5188-5327)
|
| Scan protocol |
Scanned using Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D).
|
| Description |
Gene expression after 10 hpi in resistant NIL rep 2
|
| Data processing |
Data extraction from Images was done using Feature Extraction software and Normalization of the data was done in GeneSpring GX : 75th percentile shift method .
|
| |
|
| Submission date |
Jun 29, 2022 |
| Last update date |
Nov 30, 2023 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL13636 |
| Series (1) |
|
