3 ivt express kit

Gene expression profiling was performed as previously described [17 (link)]. Reverse transcription was performed on 200 ng (2E-10 kg) of total RNA, followed by cDNA synthesis and the generation of biotin-labeled cRNA using the 3’ IVT express kit (Affymetrix, Santa Clara, CA, USA). Fragmentation of the labeled cRNA ensued and finally the fragmented and labeled cRNA was loaded on the Affymetrix GeneTitan. The hybridization mixture was loaded on Affymetrix Human_Genome_HT_HG-U133_Plus_PM GeneChip 96-well arrays. The subsequent steps (hybridization, washing and scanning) were performed within the GeneTitan. In the Affymetrix Expression Console the generated “.CEL” files were subjected to normalization employing the default settings of the RMA method. The generated data have been uploaded to the Gene Expression Omnibus data repository under the following access code: [GEO:GSE41313].
Breast cancer subtypes were determined using hierarchical clustering. Clusters were obtained with average distance linkage hierarchical clustering with non-centered correlation as distance metric. The three resulting clusters were matched to previously established intrinsic subtypes [43 (link)] of these cell lines (which were based on a different chip-type) and the clusters were labeled as ‘basal’, ‘luminal’ and ‘normal-like’.

RNA (from three biological replicates) was prepared for arrays using the 3′ IVT Express Kit (Affymetrix, USA) as per manufacturer protocol—100 ng RNA and 15 amplification cycles. Amplified RNA was given to the Boston Children’s Hospital microarray core facility for hybridization to GeneChip Human Genome U133 plus 2.0 gene expression arrays (Affymetrix, USA) for hybridization and imaging as per manufacturer protocols. Data has been submitted to the NCBI GEO database under accession GSE64536.

Gene expression profiling and analysis was performed as described in detail elsewhere 65. Briefly, 1 × 10⁷ human peripheral blood T cells from five donors were stimulated for 12 and 24 h with plate‐bound CD3/CD28 and CD3/CD63 mAbs in the presence or absence of ct‐CD45. Cell samples were lysed in 500 μL of TRIzol (Life Technologies). Following RNA isolation, the aqueous phase containing the RNA was further purified using RNeasy MinElute Cleanup Kit (Qiagen). RNA integrity was confirmed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Hundred nanograms of total RNA was used for target preparation by 3′ IVT Express kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions. Hybridization cocktails were hybridized onto Human Genome U133 Plus 2.0 GeneChips (Affymetrix). Chips were read by a GeneChip scanner 3000 (Affymetrix). Microarray data were normalized and gene expression measures derived using the RMA algorithm and the Bioconductor package ‘Affy’ (http://www.bioconductor.org).