Computational protocol: Genome scale transcriptome analysis of shoot organogenesis in Populus

Similar protocols

Protocol publication

[…] The probe-level data were normalized and summarized using the GC Robust Multichip Average (GCRMA) [] algorithm using affylmGUI []. The algorithm computes gene expression summary values for Affymetrix data in three steps: a background adjustment using sequence information, quantile normalization, and finally expression value summarization. The summary values are based on a log2 scale.Differentially expressed genes were identified by LIMMA (Linear Models for Microarray Data) [,]. LIMMA identifies differential expression via a modified t-test of gene expression between two treatments, using B statistics to rank differentially expressed genes. The BH P-value adjustment (Benjamini and Hochberg's FDR) was applied, using an adjusted P-value of 0.01 as a cutoff.To reveal both global expression changes compared to the starting explant developmental state, and the specific expression changes taking place at each stage, two sets of contrasts between time points were used. First, the expression during each of the stages was compared with the baseline explant (CIM0). Second, the expression at each stage was compared with that of the previous time point.Because of the well-established precision of the Affymetrix platform, and that our goal was to broadly cataloging patterns of gene expression, not to precisely estimate expression changes for specific genes, we did not conduct RT-PCR validation studies. Quality control studies have shown that results from RT-PCR are in agreement with microarray for genes with medium and high expression, and is not substantially more precise than the array platform employed itself []. […]

Pipeline specifications

Software tools GC–RMA, affylmGUI, limma
Application Gene expression microarray analysis
Organisms Populus alba, Populus tremula, Arabidopsis thaliana