|
| Status |
Public on Jun 20, 2017 |
| Title |
chip4_0Cu_Cy5_75Cu_Cy3 Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Seedlings
|
| Organism |
Oryza sativa |
| Characteristics |
treatment: 0µM CuSO4
variety: Nippombare
age: 8 days
|
| Treatment protocol |
Rice (Oryza sativa subsp japonica), cv. Nipponbare, seeds were surface-sterilized and stratified for 2 d at 4° C, and then germinated on ½MS plates with 1% Sucrose (Murashige and Skoog 1962). Two different Cu concentration conditions were compared, slight Cu deficiency (0 µM CuSO4) and Cu excess (75 µM CuSO4).
|
| Growth protocol |
The plants were cultured in plates during 8 days under diferent concentration of CuSO4 in the medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated with the RNeasy plant mini kit (QIAgen, Hilden, Germany; Ref. 74904), following the manufacturer’s instructions. The aRNA amplification and labeling were performed with the AminoAllyl MessageAmpTM II aRNA Amplification kit (Ambion) according to the manufacturer’s instructions. One microgram of total RNA of each sample was used for the amplification. The final concentration of obtained aRNA was determined with NanoDrop ND1000 Spectrophotometer.
|
| Label |
Cy5
|
| Label protocol |
The obtained amplified RNA (aRNA) was labeled by using the CyDye Post-Labeling Reactive Dye Pack, which generates fluorescent Cy3- and Cy5-labeled probes by a post-labeling (amino allyl) method (GE Healthcare). Each biological replicate was labeled with Cy3 and Cy5 to produce four pairs of replicate dye-swaps, which were used for microarray hybridization
|
| |
|
| Channel 2 |
| Source name |
Seedlings
|
| Organism |
Oryza sativa |
| Characteristics |
treatment: 75µM CuSO4
variety: Nippombare
age: 8 days
|
| Treatment protocol |
Rice (Oryza sativa subsp japonica), cv. Nipponbare, seeds were surface-sterilized and stratified for 2 d at 4° C, and then germinated on ½MS plates with 1% Sucrose (Murashige and Skoog 1962). Two different Cu concentration conditions were compared, slight Cu deficiency (0 µM CuSO4) and Cu excess (75 µM CuSO4).
|
| Growth protocol |
The plants were cultured in plates during 8 days under diferent concentration of CuSO4 in the medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated with the RNeasy plant mini kit (QIAgen, Hilden, Germany; Ref. 74904), following the manufacturer’s instructions. The aRNA amplification and labeling were performed with the AminoAllyl MessageAmpTM II aRNA Amplification kit (Ambion) according to the manufacturer’s instructions. One microgram of total RNA of each sample was used for the amplification. The final concentration of obtained aRNA was determined with NanoDrop ND1000 Spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
The obtained amplified RNA (aRNA) was labeled by using the CyDye Post-Labeling Reactive Dye Pack, which generates fluorescent Cy3- and Cy5-labeled probes by a post-labeling (amino allyl) method (GE Healthcare). Each biological replicate was labeled with Cy3 and Cy5 to produce four pairs of replicate dye-swaps, which were used for microarray hybridization
|
| |
|
| |
| Hybridization protocol |
From http://ag.arizona.edu/microarray/Microarraymethod1.doc Microarrays were re-hydrated by exposing them to water vapor for 10 seconds (four times). Next microarrays were cross-linked by ultraviolet (UV) irradiation of 180 mJ in a Stratalinker 1800 UV (Stratagene). Subsequently, microarrays were washed with 1% SDS (w/v) (Sodium Dodecyl Sulfate) for 5 min and 10 times with H2O milliQ (Millipore), washed with 100% ethanol 5 times and then dried by centrifugation at 200 g. After this pre-treatment, microarrays were hybridized overnight at 55 °C with hybridization solution [48 pmol of each labeled sample; deionized formamide 50% (v/v);] 3X SSC (Saline-Sodium Citrate); Denhardt´s solution [5 X and SDS 0.1% (w/v)]. The hybridization solution was denaturalized for 5 min at 65 °C and applied between the microarray and a coverslip LifterSlipTM (Erie Scientific).
|
| Scan protocol |
The hybridized microarray slides were washed and scanned using a Gene Pix Autoloader (Axon/Molecular Devices, 4200A01, Sunnyvale, CA). Spot finding and data extraction was done using GenePix Pro 6 software (Axon/Molecular Devices, Sunnyvale, CA).
|
| Data processing |
Quality control, normalization and determination of the differentially expressed genes were conducted in R using the Limma package (Smyth 2005), as previously described in (Andrés et al. 2009). To determine the false-positive ratio (FDR), the P values were adjusted by a test multiple test according to the method of Benjamini and Hochberg´s (Benjamini and Hochberg 1995). The genes that showed a differential expression with fold-change (FC) minimum of 2 for both conditions, represented as log2(FC) ≥ |1|of > 2-fold (-1 > log-fold change [½MS + 75 µM/ ½MS] > 1) with a P value of 0.05 (FDR = 0.5%) were selected.
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| |
|
| Submission date |
Oct 24, 2016 |
| Last update date |
Jun 20, 2017 |
| Contact name |
Fernando Andres |
| E-mail(s) |
andres@mpipz.mpg.de
|
| Organization name |
Max Planck Institute for Plant Breeding Research
|
| Department |
Plant Developmental Biology
|
| Lab |
Prof George Coupland
|
| Street address |
Carl von Linne 10
|
| City |
Cologne |
| ZIP/Postal code |
50829 |
| Country |
Germany |
| |
|
| Platform ID |
GPL8756 |
| Series (1) |
| GSE89109 |
Global tanscriptome analysis in Oryza sativa identifies genes regulated by copper in the medium |
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