A modified hot borate method (Wan and Wilkins, 1994) was used to extract RNA from the pooled samples. Tissue was ground in liquid nitrogen and PVP-40 (200 mg) and put into a pre-chilled collection tube with 6-8 mg proteinase K and 10 ml 80ºC XT buffer (ratio 5 ml/g). Each sample was mixed, incubated on ice for 1 hr, and centrifuged at 10,000-13,000 rpm for 20 min at 4ºC. The supernatant was filtered through sterile miracloth into a clean collection tube and 10 M LiCl was added (volume ratio 1:4). Samples were mixed and incubated on ice overnight. Samples were centrifuged at 13,000 rpm for 20 min at 4ºC, then washed three times with cold 2 M LiCl. The pellet was resuspended in 2 ml 10 mM Tris HCl, pH 7.5, by gentle vortexing and then centrifuged at 13,000 rpm for 10 min at 4ºC. The supernatant was decanted, 200 μl (1/10 vol) K-acetate (pH 5.5) was added, and the sample was incubated on ice for 15 minutes. Samples were centrifuged at 13,000 rpm for 10 min at 4ºC, the supernatant collected, and 5 ml 95% EtOH was added. Tubes were mixed and incubated at -20ºC overnight, then centrifuged at 13,000 rpm for 30 min at 4ºC. The pellet was washed with 4 ml 70 % EtOH, centrifuged at 13,000 rpm for 5 min at 4ºC, dried at room temperature, and resuspended in suitable volume of DEPC-dH2O. Quantitative and qualitative determination of RNA was performed with gel electrophoresis.
Label
biotin
Label protocol
RNA labeling was performed according to instructions in the GeneChip One Cycle Target Labeling Kit (Affymetrix, Santa Clara, CA, USA).
Hybridization protocol
Standard Affymetrix protocols were followed.
Scan protocol
Used Affymetrix 3000 GeneChip Scanner, and standard Affymetrix protocols were followed.
Description
sample from a pool of eight fruits from transgenic line DefH9-iaaM
Data processing
Standard Affymetrix protocol was followed for image processing. For statistical analyses, R statistical software was used. We used the RMA method (Irizarry et al., 2003) for background subtraction and normalization to preprocess raw probe data and produce the gene expression matrix. To determine which genes were differentially expressed among different groups, one-way ANOVA was used to obtain a p-value for each gene. Then all p-values were BH adjusted (Benjamini and Hochberg, 2000) for multiple hypotheses. Genes with adjusted p-values < 0.05 were considered differentially expressed in different groups. We used R package LMGene (Rocke and Durbin, 2003; Rocke, 2004) to perform one-way ANOVA and R package mulltest (Pollard and van der Laan, 2004) to adjust multiple hypotheses.
