Subsequently and with the use of the TM4 software selleckbio suite, the obtained spot values were corrected for background fluorescence and inconsistent hybridization results across dye swap replicates. The data were log2 transformed and LOWESS normalized correcting for pin induced spot intensity biases. To verify reproducibil ity between spots across slides, F tests were performed applying a 95% confidence threshold and allowing removal of inconsistent hybridization results. A mixed model ANOVA was used to assess the significance of the difference in expression of each gene among geno types using a false discovery rate significance threshold of 0. 05. With the multiple steps required to carry out a successful microarray experiment, it is not unusual to have noisy data.

To extract reliable infor mation from the data, non biologically significant sources of signal variation were identified and their effects removed. The following gene model was used to identify genes that were differentially expressed, Yijkl denotes the transformed intensity for a gene, u denotes the average intensity and ��ijklm captures the ran dom errors. The variation due to microarray slide used was designated as random effect, whereas, varia tions due to RNA fluorescent labeling, biological sample RNA and endosperm genotype were treated as fixed effects. Only the main effects interacting with Treatment were included in the model. Quantitative Real Time PCR 1 ug of mRNA was reverse transcribed by mixing with 1 ul of oligo dT18, 1 ul of dNTP mix, 4 ul of first strand buffer, 2 ul of 0.

1 M DTT, 1 ul of M MLV Reverse Transcriptase, and 13 ul of distilled sterile water. After reverse transcription at 37 C for 1 hour, the cDNAs were tested on a 0. 8% agarose gel and diluted to a final volume of 500 ul with distilled sterile water. PCR reaction mixtures were assembled combining, 2 ul of diluted cDNA, 2 ul of gene specific forward primer, 2 ul of gene specific reverse primer, 5 ul of 10x reaction buffer, 2 ul of 50 mM MgCl2, 2. 5 ul of 2 mM dNTP mix, 5 ul of diluted SYBR Green, 0. 5 fluorescein, 0. 2 ul of Platinum Taq DNA polymerase. Real Time amplification was performed using an iCycler using the following thermal cycling profile, 95 C for 5 min followed by 50 cycles of 95 C 30 sec, 55 C 30 sec, 72 C 30 sec. All reactions were performed in triplicate.

The obtained threshold cycles were averaged across replicates and sample errors computed. Ratios of CT values were computed and used to corroborate the observed GSK-3 hybridization pat terns. Linear regression analyses showed a strong corre lation between measurements of gene expression assessed by microarrays and by qRT PCR, with correla tion coefficient r2 0. 83. Gene specific primers were selected and designed from sequences near the 3 end of the gene using the Zeastar Unigene sequence database.