|
| Status |
Public on Mar 10, 2016 |
| Title |
Oryza latifolia-120mM_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Whole seedling, 120mM NaCl, replicate 2
|
| Organism |
Oryza sativa |
| Characteristics |
organism (germplasm accession): Oryza sativa (IRGC 100965)
tissue: whole seedling
age: 16 days after emergence
stress level: 120 mM NaCl
stress duration: 48 hrs
|
| Treatment protocol |
Seedlings in flasks were challenged with 0 & 120 mM NaCl in Yoshida nutrient medium (with additional CaCl2 added at a molar concentration ratio of 1:6) with three replicates per treatment. Half strength salt stress was applied at 13 d after emergence and after another 24 h the full strength stress was applied. The pH of the nutrient solution was maintained at 5.0 on a daily basis and the solution was renewed every week.
|
| Growth protocol |
Seeds were heat treated at 48°C for 5 d, then sterilized with 1% sodium hypochlorite and incubated on pre-soaked filter paper in a sterile petridish at 20°C for 48 h. Germinated seeds were transferred to test tubes filled with Yoshida nutrient medium (Yoshida et al., 1976) for seedling establishment for 7 d and then the seedlings were transferred to aluminum foil wrapped 250 mL conical flasks. Individual seedlings were held in place using a sponge bung in each flask and roots were suspended in nutrient solution. Flasks were maintained in a growth room with 16/8 h photoperiod, 28/20°C day/night temperature, 70-75% relative humidity and average 290 μmol m-2 s-1 photosynthetic photon flux.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extractions were carried out using the QIAGEN RNeasy Plant Mini Kit from the 16 day old fresh whole seedlings (including root and shoot). For seedlings weighing more than the recommended starting material (100 mg), representative sections (tip, middle and basal portion) of root, stem and leaves of each seedling were kept using sterile scissors. RNA was quantified using a Nanodrop ND-1000 VIS spectrophotometer (v. 3.2.1) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 300 ng RNA using the ‘One color - Low Input Quick Amplification Labeling Protocol v.6.5, May 2010’ (available from www.agilent.com/chem/dnamanuals-protocols) according to the manufacturer’s (Agilent) instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). The incorporation of Cy3 dye and the yield of cRNA were checked with the NanoDrop ND-1000 VIS spectrophotometer (v. 3.2.1).
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| |
|
| Hybridization protocol |
Cy3-labelled cRNA (1.65 ug) was fragmented at 60°C for 30 minutes by 11.0 µL of 10x blocks and 2.2 µL of fragmentation buffer in a reaction volume of 55 uL as per the manufacturer’s instructions. After that, 55 µL 2 x GE hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Rice Gene Expression 4x44K Microarray (product G2519F; Design ID-015241) for 17 h at 650C in an Agilent hybridization rotisserie. Microarrays were then washed for 1 minute at room temperature with GE Wash Buffer 1 (Agilent), for 1 minute with GE Wash buffer 2 (Agilent), for 10 s with 100% acetonitrile followed by another wash in a stabilising and drying solution for 30 s.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides at ‘Profile AgilentG3_GX_1Color’.
|
| Description |
Gene expression in 120mM NaCl stressed (for 48 hrs)_16 day old_whole rice seedling
|
| Data processing |
Data were acquired from the scanned images using Agilent feature extraction software version 9.5.3 to obtain background subtracted and spatially detrended Processed Signal intensities. After the successful completion of extraction, the QC report for each extraction set was critically evaluated and the features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Mar 09, 2016 |
| Last update date |
Mar 10, 2016 |
| Contact name |
Mohammad Rashed Hossain |
| Organization name |
Bangladesh Agricultural University
|
| Department |
Genetics and Plant Breeding
|
| Street address |
Factulty of Agriculture (East Building)
|
| City |
Mymensingh |
| ZIP/Postal code |
2202 |
| Country |
Bangladesh |
| |
|
| Platform ID |
GPL8852 |
| Series (1) |
| GSE79043 |
Salinity induced gene expression profiling in 8 diverse rice genotypes |
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