Microarray data for Ito et al. (2004)
I. Experiment
1. Experimental design
Gene expression changes in Arabidopsis inflorescences were analyzed after posttranslational activation of an AG-GR fusion protein. To this end, inflorescences of ag-1
35S::AG-GR plants were treated with dexamethasone or a mock solution for different
durations and probes derived from dexamethasone or mock-treated samples were cohybridized to a cDNA array that is enriched for flower-specific transcripts.
2. Samples used, extract preparation and labeling
Inflorescences of ag-1 35S::AG-GR plants were treated with dexamethasone or a mock
solution for different durations (0, 4, 8, 10, or 16 hours) and floral buds of stages 1-10
were collected for the experiments. Two sets of samples were collected from independent
transgenic lines that showed similar phenotypic responses to AG-GR activation. Total
RNA was isolated from these samples using the Qiagen RNeasy kit (Qiagen, Valencia,
CA, USA). The probe labeling protocol was described previously (Wellmer et al., 2004).
For set 1, dexamethasone-treated samples were labeled with Cy3 and mock-treated
samples with Cy5. For set 2, the dyes used for labeling of the co-hybridized samples were
switched to avoid dye-related artefacts.
3. Hybridization procedures and parameters
The hybridization protocol was described previously (Wellmer et al., 2004).
4. Measurment data and specifications
Analysis of microarray data was performed as follows. Scanning of slides and
quantitation of signal intensities was performed as described (Wellmer et al., 2004),
using an Axon GenePix 4000A scanner and the associated GenePix Pro 4.1 software
(Axon Instruments, Union City, CA). The GenePix .gpr files were processed in the
Rosetta Resolver Gene Expression Data Analysis System (Rosetta Biosoftware, Kirkland,
WA). Processing in this system consists of error correction and calculation of a P-value
of differential expression using the intensity-error estimation from the .gpr file. Error
correction consists of a simplified version of the algorithm described by Schadt et al.
(2001), in which a piece-wise linear function replaces smoothing splines to fit and correct
intensity non-linearity. Calculation of P-values consists of a statistic that combines
additive and multiplicative error components in both channels of a two-colored
experiment. The resultant ratio profiles (that is, two-channel, error processed microarray
scans) were combined into ratio experiments in the Resolver system as described in
Stoughton and Dai (2002). Within the Resolver system, we performed the analysis at the
so-called sequence level, i.e., when there are multiple datapoints for the same gene in the
same hybridization (as a result of reporter or probe sequences from that gene appearing
more than once on the microarray; see Wellmer et al. (2004)) such data are combined.
When combining data, it is assumed that the ratio measurement with the lowest overall
error is closest to the true value for that sequence, and weights are constructed such that
the feature or reporter with the lowest error is given the greatest weight (Stoughton and
Dai, 2002).
Raw data for the two sets of biologically independent samples are provided in the folder
‘raw data’. The results of five time points (hybridizations) per set are given. The data
files are designated for example ‘0h_1.gpr’, which is the 0 hour time point of set 1.
Raw data were loaded into Resolver and processed as described above. The processed
data are listed in the folder ‘processed data’. The data files are designated for example
‘AG0h1.xls’, which is the 0 hour time point of set 1. Columns contain the following
information:
Reporter Name: unique identifier of a given array element
Intensity1: Absolute intensity measurement for the Cy3-labeled sample after background subtraction and
normalization
Intensity2: Absolute intensity measurement for the Cy5-labeled sample after background subtraction and
normalization
P-value: Value describing the confidence that the element's expression ratio reflects a change in expression
not due to measurement errors
Log(Ratio): Log10 ratio (Intensity2 divided by Intensity1)
Flagged?: Indicates whether data from the array element were marked as failed during quality control
procedures
The data of the replicate experiments were then combined and analyzed in Resolver as
outlined above. The data files for the five time points are provided in the folder ‘Time
Course’ and are designated for example ‘AG0h.xls’, which is the Resolver output file for
the 0 hour time point. Columns contain the following information:
Sequence Code: Column contains gene identifiers or unique identifiers (in case an element was not
sequenced)
Intensity1: Absolute intensity measurement for channel1 after background subtraction and normalization
Intensity2: Absolute intensity measurement for channel2 after background subtraction and normalization
Log(Ratio): Log10 ratio (Intensity2 divided by Intensity1)
Log(Error): Displays the error in the log ratio
X Dev: The difference between the experiment and baseline intensity, divided by the standard deviation of
the difference.
P-value: Value describing the confidence that the element's expression ratio reflects a change in expression
not due to measurement errors
Flagged?: Indicates whether data from the array element were marked as failed during quality control
procedures
II. Array design
The design of the cDNA array used in this study was described previously (Wellmer et
al., 2004).
III. References
Schadt, E.E., Li, C., Ellis, B., Wong, W.H. (2001). Feature extraction and normalization
algorithm for high-density oligonucleotide gene expression array data. J. Cell. Biochem. 84 (S37),
120-125.
Stoughton, R.S. and Dai, H. (2002). Statistical combining of cell expression profiles, US Patent
No. 6,351,712.
Wellmer, F., Riechmann, J.L., Alves-Ferreira, M. and Meyerowitz, E.M. (2004) Genomewide analysis of spatial gene expression in Arabidopsis flowers. Plant Cell 16, 1314-1326.