normalization data transformation protocol
Background subtraction, quantile normalization, and summarizing probe sets from Affymetrix expression microarrays raw CEL files were done with Affymetrix Power Tools (APT) software. HuEx-1_0-st-v2.r2.pgf HuEx-1_0-stv2. na30.hg19.probeset.csv ID_REF = VALUE = Quantile normalized gene level expression values from Affymetrix Power Tools (APT) software.
array scanning protocol
After washing and staining of hybridized arrays with an Affymetrix Fluidics Station, arrays were scanned on Gene Array Scanner 2500 (Affymetrix) to capture the raw probe signal intensities in CEL files.
Samples were hybridized with GeneChip Human Exon 1.0 ST Arrays (Affymetrix) in 45ºC hybridization oven at 60rpm for 18 hours.
Single-stranded cDNA was generated from the amplified cRNA with the Ambion® WT Expression Kit and then fragmented and labeled with the WT Terminal Labeling Kit (Affymetrix).
nucleic acid extraction protocol
RNA was isolated from transfected cells post 24 hours of transfection with the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
HCT116 (p53-/-) cell lines were grown in DMEM supplemented with 10% fetal bovine serum and cultured at 37ºC incubator.
sample treatment protocol
HCT116 (p53-/-) cell lines were transiently transfected with pcDNA3.1 control plasmid, pCMV-p53WT, or pCMV-p53-mutants (R175H, C176F, G245D, R273C, R280T, and R282W) with Lipofectamine 2000 (Life Technologies) according to standard protocol.