Computational protocol: Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants

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Protocol publication

[…] Raw CEL data was generated using MAS 5.0 (Affymetrix). An RMA measure of gene expression was calculated using the affylmGUI (affy and limma) package implemented for the Bioconductor project running in the R environment [,,]. Perfect match values only were used with quantile normalization []. Raw CEL files were also loaded into dchip (v1.3 release date: 07/20/2005) and were normalized and modeled using the PM-MM and PM-only algorithms. Scatter plots of the log2 expression measures between a set of replicates processed with different methods and Pearson correlation coefficients were prepared using Excel 2003 (Microsoft, Redmond, WA).For the statistical tests, affylmGUI was used to compute the moderated t-statistic [,], and the log fold changes. P-values were adjusted for multiple testing using the Storey q-value method []. For dchip, the PM-only expression values of sets of replicates were used to compare with other genotypes using the 'compare samples' function, with the following criteria: means separated by at least 20 and a fold change of 1.2 (using the lower bound of 90% confidence interval). SOM cluster analysis was carried out in GeneCluster 2.1.7 (Broad Institute), chosen as the number of likely patterns was low and it was not important to identify sub-clusters. The 12 clusters that result from SOM clustering of 800 inner integument genes are shown in additional file .Genes were considered putatively absent in a sample if the average value was below 12, which was chosen by assessing the values of a set of putatively root specific genes [] in the pistil samples (additional file ). [...] The RNA samples for RT-PCR were subjected to DNase treatment (Promega, Madison, WI) and digestion by two four-base cutter restriction enzymes to ensure complete digestion of any contaminating DNA. Two of the three biological replicates used for the microarrays were used in the reverse transcription and quantitative PCR. 1 μg of each RNA was used in reverse transcription reactions with either 3.75 units of Thermoscript (Invitrogen, Carlsbad, CA) or no reverse transcriptase as a -RT control, which was tested for contaminating genomic DNA in PCR reactions. 2 μl of a 1:40 dilution of the RT reactions were used in each quantitative PCR (qPCR) reaction. Primers were designed using the SYBR Green option of the Beacon 2.0 primer design software (Premier Biosoft, Palo Alto, CA) (Additional file ). qPCR reactions were carried out using an iCycler (Biorad, Hercules, CA) and the following PCR reaction mix: 20 mM Tris pH 8.4, 50 mM KCL, 3 mM MgCl2, 4% glycerol, 20 nM fluorescein diacetate, 0.5× BSA (New England Biolabs, Beverly, MA), 1:50 000 diluted SYBR GREEN I (Cambrex Bio Science, Rockland, ME), 0.2 mM each dNTP, 0.24 μM each primer and 0.6 U iTaq (Biorad, Hercules, CA).The fluorescence threshold at which the cycle number (Ct) was calculated was set at 25 CF RFU (curve-fit relative fluorescence units) for all experiments, close to the automatically determined threshold for each plate. The 60S ribosomal protein RPL14B gene (At4g27090) was used as a reference and showed very similar Ct values (range: 19.17 – 19.70) in all sample types tested. The relative starting quantity of cDNA for a particular gene was determined in GENEX (Biorad) using the following equation: relative quantity = efficiency (control Ct-experimental Ct) based on Livak [] and Vandesompele []. The mean PCR efficiencies of the primer sets were determined using LinRegPCR [], using a linear regression model. […]

Pipeline specifications

Software tools affylmGUI, limma, GeneCluster, LinRegPCR
Applications Gene expression microarray analysis, qPCR
Organisms Arabidopsis thaliana