|
| Status |
Public on Apr 23, 2009 |
| Title |
leaf_3% light_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Uros III mutant grown 3% light
|
| Organism |
Hordeum vulgare |
| Characteristics |
Barley cultivar Golden promise Uros III mutant
|
| Growth protocol |
Barley plants were grown under glasshouse conditions with a 16 hours light, 21oC /8 hours dark, 16oC growth regime. Plants grown under 30% and 3% light were grown under 1 or 3 layers of 70% shade cloth, respectively. Light titration experiments were undertaken in growth cabinets with incident light (umol m-2 s-1) measured at the soil surface.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from leaf tissue using TRIzol reagent (Gibco BRL) and precipitated with isopropanol then resuspended in water.
|
| Label |
Cy5
|
| Label protocol |
For the labelling procedure, 50 µg of total RNA was used for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), following the two-step labelling method of Schenk et al. (2000) Proc.Natl Acad Sci USA 97:11655–11660.
|
| |
|
| Channel 2 |
| Source name |
Wild type grown 3% light
|
| Organism |
Hordeum vulgare |
| Characteristics |
Barley cultivar Golden promise
|
| Growth protocol |
Barley plants were grown under glasshouse conditions with a 16 hours light, 21oC /8 hours dark, 16oC growth regime. Plants grown under 30% and 3% light were grown under 1 or 3 layers of 70% shade cloth, respectively. Light titration experiments were undertaken in growth cabinets with incident light (umol m-2 s-1) measured at the soil surface.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from leaf tissue using TRIzol reagent (Gibco BRL) and precipitated with isopropanol then resuspended in water.
|
| Label |
Cy3
|
| Label protocol |
For the labelling procedure, 50 µg of total RNA was used for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), following the two-step labelling method of Schenk et al. (2000) Proc.Natl Acad Sci USA 97:11655–11660.
|
| |
|
| |
| Hybridization protocol |
The pre-hybridisation of the microarray slides, hybridisation of the target cDNA and subsequent washing of the slides to remove unbound target was performed following the protocol for the CMT-GAPS-coated microscope slides provided by the manufacturer (Corning, USA).
|
| Scan protocol |
The slides were scanned with a GenePix 4000 A microarray scanner (Axon Instruments, Union, Calif.). The features were analysed using the GENEPIX PRO 4 software, and unsatisfactorily segmented features were either manually adjusted or discarded to ensure the integrity of the data obtained.
|
| Description |
none
|
| Data processing |
The analysis of the microarray data files was carried out using functions contained in the TRMA statistical pack- age. These functions operate as part of a statistical software package called R (http://www.r-project.org/). A detailed description of TRMA (tools for R microarray analysis; Wilson et al. 2003) is available online (http:// www.csiro.au/gena/trma). The data sets generated from the GENEPIX software were loaded into TRMA using the LOADGENEPIXFILE function. Normalisation of log2 ratio values was performed using the SPATIALLYNORMALISE function. This method of normalisation corrected for spatial and intensity-dependent effects of fluorescence across the microarray slide (Wilson et al. 2003). In addition, the possible biases in fluorescence due to differences in the efficiency of incorporation of the two dyes and unequal loading of cDNA samples were also corrected. Using the median values of the normalised log ratios for each gene in each replicate, we determined differentially expressed genes using the FINDDIFFEXPGENES function. Differentially expressed genes are selected as outliers in a Gaussian distribution of the entire data set. Therefore, a ratio cut-off was empirically computed from the normalised log2 ratio data, which were rescaled and centred in order to make direct comparisons between replicates. The COMPAREINTERESTINGGENES function was used to compare the differentially expressed genes among the replicates. The stringency was set to 100%—i.e. only those genes differentially expressed in all of the replicates tested, with a consistent direction of expression were selected.
|
| |
|
| Submission date |
Jun 12, 2008 |
| Last update date |
Apr 23, 2009 |
| Contact name |
Andrew Spriggs |
| E-mail(s) |
andrew.spriggs@csiro.au
|
| Phone |
612-6246-5193
|
| Organization name |
CSIRO Plant Industry
|
| Department |
Bioinformatics Group
|
| Street address |
GPO Box 1600
|
| City |
Canberra |
| State/province |
ACT |
| ZIP/Postal code |
2601 |
| Country |
Australia |
| |
|
| Platform ID |
GPL6956 |
| Series (1) |
| GSE11776 |
Suppressing the barley uroporphyrinogen gene by Ds activation tagging element generates developmental photosensitivity |
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