7 protocols
VALUE = log2 transformed RMA signal value
The arrays were scanned with an Affymetrix Genechip Scanner 3000 and processed automatically into digital CEL files.
The raw data (CEL files) were analyzed with Affymetrix Gene Expression Console. Background correction and normalization were conducted using the RMA algorithm implemented in the Affymetrix package (version 1.26.1) of the Bioconductor software.
The RNA was isolated using TriZol according to Invitrogen's protocols as directed by the Affymettrix GeneChip Expression Analysis Technical Manual.
The biotinylated cRNA targets were cleaned up, fragmented, and hybridized to GeneChip expression arrays at 45˚C for 16 hours in a rotation oven. The hybridized chips were subjected to washes and stained in a Fluidics Station 450.
MCF10A cells were grown in 2D and 3D cells as described previously (Xiang and Muthuswamy 2006, PMID:16472698).
5μg total RNA was reverse transcribed using a T7- Oligo(dT) promoter primer for first-strand cDNA synthesis. Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA was purified and served as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling.