Aliquots of total RNA of 149 frozen tumor samples was available for this study, for 13 samples (8 out of 78 and 5 of the 145 tumor series, see above) new RNA was isolated from available frozen tumor tissue as described previously [6 (link),13 (link),22 (link)]. Two-hundred nanogram total RNA was amplified using the Low RNA Input Fluorescent Labeling Kit (Agilent Technologies). Cyanine 3-CTP or Cyanine 5-CTP (Perkin Elmer) was directly incorporated into the cRNA during in vitro transcription. A total of 200 ng of Cyanine-labeled RNA was co-hybridized with a standard reference to custom 8-pack mini-microarrays (MammaPrint, Agendia) at 60°C for 17 hrs and subsequently washed according to the Agilent standard hybridization protocol (Agilent Oligo Microarray Kit, Agilent Technologies). The reference sample consisted of pooled and amplified RNA of 105 primary breast tumors selected from patients of the clinical validation series [13 (link)] in such a way that it had a similar proportional distribution between good and poor profile patients. Sufficient reference material was generated for over 30,000 hybridizations. For each tumor two hybridizations were performed by using a reversal fluorescent dye.
The customized mini-array contained 1,900 60-mer oligonucleotide probes that comprise the 232 prognosis related genes [6 (link)] identical to the probes on the original array, including the genes of the 70-gene prognosis classifier, spotted in triplicate. Each array additionally includes 289 probes for hybridization and printing quality control as well as 915 normalization genes. Eight identical MammaPrint arrays are present on a single 1" × 3" slide, allowing for eight individual hybridizations to be performed simultaneously. After hybridization the slides were washed and subsequently scanned with a dual laser scanner (Agilent Technologies). Microarray raw data are available at the European Bioinformatics Institute (EBI) Arrayexpress database;[23 (link)] accession number E-TABM-115.
The customized mini-array contained 1,900 60-mer oligonucleotide probes that comprise the 232 prognosis related genes [6 (link)] identical to the probes on the original array, including the genes of the 70-gene prognosis classifier, spotted in triplicate. Each array additionally includes 289 probes for hybridization and printing quality control as well as 915 normalization genes. Eight identical MammaPrint arrays are present on a single 1" × 3" slide, allowing for eight individual hybridizations to be performed simultaneously. After hybridization the slides were washed and subsequently scanned with a dual laser scanner (Agilent Technologies). Microarray raw data are available at the European Bioinformatics Institute (EBI) Arrayexpress database;[23 (link)] accession number E-TABM-115.
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