Fibroblast explant cells from a Li-Fraumeni patient
Extracted molecule
polyA RNA
Extraction protocol
Extracted with oligo-dt cellulose using a Micro FastTrack Kit (Invitrogen)
Label
Biotin-labeled cRNA were stained with streptavidin phycoerythrin stain solution. Signal was amplified with goat anti-streptavidin antibody and biotinylated goat IgG (Affymetrix).
Label protocol
We synthesized biotin-labeled cRNA with an ENZO RNA transcript labeling kit using 0.7 µg cDNA per reaction. cRNAs were purified with RNeasy purification columns (Qiagen).
Hybridization protocol
cRNA, 15 µg per sample, was heated at 80ºC for 33 min in fragmentation buffer. Fragmented cRNAs were hybridized to U95Av2 GeneChips (Affymetrix) for 16 hr at 45ºC.
Scan protocol
We scanned stained GeneChips on an Agilent GeneArray Scanner (Agilent Technologies)
