The G1E erythoid model system was profiled using the MG-U74Av2 Affymetrix oligonucleotide microarray. These cells are induced to differential along the erythroid lineage by functional restoration of GATA-1. G1E cells are derived from GATA-1 null embryonic stem cells, and this experiment employed a clone containing a fusion construct combining the estrogen receptor ligand binding domain with GATA-1. When estradiol is added to the culture medium, GATA-1 dissociates from heat shock proteins and is available in the nucleus as a transcription factor. We collected RNA at 3, 7, 14, 21, and 30 hours after addition of estradiol. During this period, the cells underwhen phenotypic erythroid development. We believe that the 30 hour time course of this experiment represents red cell differentation from the late CFU-E stage through the basophilic erythroblast stage. The experiment was repeated three times, therefore 18 microarrays were used in total.
The chips were analyzed in MAS 5.0 (Affymetrix Microarray Suite software), including absolute analyses and comparative analyses between time zero and each later time point, as well as comparative analyses between consecutive time points. Both raw data and summary data are available for download, in both Microsoft Excel and tab-delimited text format.
Both sets of data include identifying information in the first several columns, followed by the absolute analyses and the comparative analyses between consecutive time points, and between baseline and each time point. The summarized data combine the three replicates into average values, as below. The spreadsheets use the "Wingdings 3" font to indicate changes as graphic symbols. In the text versions of the results, or if the Wingdings font is not available, these characters will be represented as letters. The table given below shows the equivalencies. These graphic characters were used to make the display more intuitive and because a single-letter code was more helpful for automated processing
Experiments were designated "a", "b", and "c", and these letters are appended to category headings. Absolute analysis headings like 0abs_a, 0abscall_a, 0abs_p_a indicate the absolute analysis of chip "a" at baseline (zero time point). The columns list the signal value, affymetrix presence/absence call, and the "p" value for the call. On the Excel version of the spreadsheet, these values are separated from the Comparative analysis by a heavy vertical line. The next columns are of the form 7_3_slr_a, 7_3_change, 7_3_p_a. These correspond to the comparative analysis between consecutive time points of each experiment. In this example, these columns relate to the comparison of the 7 hour array to the 3 hour array within experiment "a". The columns represent the signal log ratio, the affymetrix change call (increased/decreased/not changed) and the p-value associated with the call. Another heavy vertical line separates these columns from the final set of columns on the spreadsheet: the comparative analysis versus time zero. These columns follow the same format as the previous set, except all of the time points are compared back to the time zero array.
To condense the data, triplicates were pooled by averaging values. The SLR values were linearized, averaged and log-transformed again. The "average p-value" is given, but is not a real statistic -- it is included merely to give a sense of significance.
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