RNA preparation and hybridization protocol description

 MCF7 and MCF10A were labelled and hybridized in triplicate to HG U133 Plus2 arrays and Human Exon 1.0ST arrays. MCF7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (v/v) foetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA), and MCF10A was grown in DMEM/F12 with 5% (v/v) horse serum (Invitrogen, Paisley, UK), 2 ng/mL epidermal growth factor (PeproTech, Rocky Hill, NJ, USA), 0.5 g/mL hydrocortisone, 0.5 g/mL cholera toxin, and 5 g/mL insulin (Sigma, St. Louis, MO, USA).

Exon arrayLabelling of samples for hybridisation to the newer
Exon array was performed strictly according to the GeneChip Whole Transcript
(WT) Sense Target Labelling Assay. This assay differs significantly from the
standard labelling assay in several ways. Double stranded cDNA is synthesized
using random hexamers incorporating a T7 promoter sequence. The use
of random hexamers avoids bias towards the 3’ end of transcripts; however a
drawback of this approach is that the ribosomal RNA will also be primed by the
random hexamers, possibly leading to non-specific hybridisation on the array.
Therefore a ribosomal reduction step is recommended prior to labelling. Double
stranded cDNA is used to generate cRNA in the same manner as in the standard
IVT reaction, following this random primers are again used to generate single
stranded cDNA in the sense direction for hybridisation to the array. In contrast
to plus2 arrays, it is DNA rather than RNA which is finally hybridised to
the array. Hybridisation was performed at 45C overnight, washing and staining
was performed using an FS450 automated fluidics station with fluidics protocol
FS450 0001. Scanning was carried out using the GCS3000 scanner with a 7G
upgrade. Full details of this protocol are given in the Affymetrix user manual:
GeneChip Whole Transcript (WT) Sense Target Labelling Assay Manual.
As a result 6 CEL files were created.