two probes to be compared were combined and purified (Qiagen PCR purification kit; Hilden, Germany), mixed in 3X SSC and supplemented with 20 µg poly(A) and 0.15% v/v SDS, and competitively hybridized to the B. tabaci microarray in a 65°C water bath for 16-18 h. Each microarray slide contained 5772 printed PCR products, each representing one EST. The slides were washed sequentially (2 min per wash) at ambient temperature in 1.14X SSC supplemented with 0.0285% SDS, then 1.14X SSC, 0.228X SSC and 0.057X SSC. Immediately after washing, arrays were spun dry at 1,000g for 5 min in a table-top centrifuge. The slides were scanned using an Agilent microarray scanner (Agilent Technologies, Santa Clara, CA, USA) to detect Cy3 and Cy5 fluorescence.
Scan protocol
The slides were scanned using an Agilent microarray scanner (Agilent Technologies, Santa Clara, CA, USA) to detect Cy3 and Cy5 fluorescence.
Description
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Data processing
Fluorescence intensity was quantified using Genepix Pro v6 (Axon Instruments, Union City, CA, USA). After excluding all bad hybridizing spots by visual assessment, the real intensity value of each spot was collected. Data were normalized and corrected to optimal microarray platform by taking into account local and global background intensities. The normalization method consisted of a calculation based on the ratio of medians from GenePix Pro microarray analysis software. This normalization method is based on removal of all gene-expression values higher than 15-fold and then calculating the average ratio of medians and multiplying of each value by the resulting correction factor (normalization factor medians), such that the average of the corrected ratios equals 1.0. The experiments were performed in triplicate and were reproducible, with Pearson correlation coefficients between repeat experiments for the genes that passed the significance test used (see below) of 0.86. All subsequent analyses were performed using the normalized ratio of medians as the value of gene expression, following log base 2 transformation. The ratio of medians of 25ºC-exposed and 40ºC-exposed whitefly transcripts were obtained for each gene.
