Dissected skin was minced, and placed in RNAlater (Applied Biosystems, Austin, TX) overnight at 4°C. Tissue samples were kept at -80°C, until further processing within 48 hrs. Frozen samples were lysed in RLT Buffer from an RNeasy Mini Kit (Qiagen, Valencia, CA). Samples were frozen at -80°C for storage and then total RNA was isolated according to manufacturer’s protocol (Qiagen) including the optional DNase treatment step. Quality was assessed using an Agilent 2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Following reverse transcription with an Oligo(dT)-T7 primer (Affymetrix, Santa Clara, CA), double stranded cDNA was synthesized with the GeneChip® Expression 3’-Amplification One-Cycle kit (Affymetrix, Santa Clara, CA). In an in vitro transcription (IVT) reaction with T7 RNA polymerase, the cDNA was linearly amplified and labeled with biotinylated nucleotides (Affymetrix).
Hybridization protocol
Ten micrograms of biotin-labeled and fragmented cRNA was hybridized onto MOE430v2.0 GeneChip arrays (Affymetrix) for 16 hrs at 45°C. Post-hybridization staining and washing were performed according to manufacturer’s protocols using the Fluidics Station 450 instrument (Affymetrix). Finally, the arrays were scanned with a GeneChipTM Scanner 3000 laser confocal slide scanner.
Scan protocol
The arrays were scanned with a GeneChipTM Scanner 3000 laser confocal slide scanner.
Description
mutant
Data processing
Average signal intensities for each probe set within arrays were calculated by the rma function provided within the Affymetrix package for R using a custom CDF file (Dai et al., 2005). The RMA method incorporates convolution background correction and summarization based on a multi-array model fit robustly using the median polish algorithm. Data was quantile-normalized to bring arrays onto a common scale. For this experiment, one pair-wise comparison was used to statistically resolve gene expression differences between AP groups using the R/MAANOVA analysis package (Wu et al., 2003). Specifically, differentially expressed genes were detected by using Fs, a modified F-statistic incorporating shrinkage estimates of variance components from within the R/MAANOVA package (Cui et al., 2005; Wu et al., 2003). Statistical significance levels of the pairwise comparison were calculated by permutation analysis (1000 permutations) and adjusted for multiple testing using the false discovery rate (FDR), q-value, method (Storey, 2002). Differentially expressed genes were declared at an FDR q-value threshold of 0.05. References: - Cui X, Hwang JT, Qiu J, Blades NJ, Churchill GA (2005) Improved statistical tests for differential gene expression by shrinking variance components estimates. Biostatistics 6:59-75. - Dai M, Wang P, Boyd A, Kostov G, Athey B, Jones E, et al. (2005) Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Research 33. - Storey J (2002) A direct approach to false discovery rates. Journal of the Royal Statistical Society Series B:479-98. - Wu H, Kerr M, Cui X, Churchill G (2003) MAANOVA: A software package for the analysis of spotted cDNA microarray experiments. In: The analysis of gene expression data, pp. 313-341 (Springer, New York, NY).
