tissue: primary breast tumor sample source: Guy's Hospital er (0: negative): 0 pr (0: negative): 0 her2 (1: positive, 0: negative or equivocal): 0 time to distant recurrence (years): 5.824657534 distant recurrence indicator (1=yes, 0=no): 0 sbr grade: 9 number positive nodes: 1 tumor size (cm): 3.2
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from 10 micron FFPE sections using a modified commercially available isolation kit (Zymo Research). The FFPE sections were digested with proteinase K for 18-24 hours at 55°C, spun down and the supernatant treated with a mixture of 100% ethanol and a GuSCN-based extraction buffer. The extracted material was purified on Zymo-Spin II columns, eluted with TE buffer and the RNA reverse transcribed into cDNA using random hexamers and the High Capacity cDNA kit (Life Technologies).
Label
Multiplexed - NED, VIC, Quasar 670, 6FAM
Label protocol
The probe for each gene within a multiplex is labeled with a unique fluorophore with the exception of the two reference genes, which were both labeled with NED in the same mix. Amplifications were performed with AmpliTaq Gold in a buffer containing 15 mM Tris-HCl, 50 mM KCl, pH 8.0 , 2.5 mM MgCl2, 200 uM dAGC, 400 uM dUTP, and uracil-N-glycolysis.
Hybridization protocol
n/a
Scan protocol
n/a
Description
FFPE samples from chemotherapy-naïve hormone-receptor-negative breast cancer annotated with distant recurrence information.
Data processing
CT values for target genes are non-detected if there is a lack of gene amplification within 40 thermocycles. ddCT values for target genes are generated by first normalizing to two reference genes (PPIG and NUP214), then to a universal human reference RNA (Stratagene) that was amplified with the same genes. Samples assayed in the same batch are normalized to a corresponding RNA reference; altogether, six RNA references were employed. CPMC samples are normalized to RP_1. Guy's Hospital samples: 5037-057, 5037-228, 5040-026, 5040-042, 5040-047, 5040-057, 5040-058, 5040-060, 5040-087, 5040-093, 5040-100, 5040-103, 5040-112, 5040-154, 5040-173, 5040-178, 5040-193, 5040-197, 5040-253, 5040-255 are normalized to RP_2, the remaining Guy's hospital samples are normalized to RP_3, Mayo clinic samples: Mayo 06, Mayo 15, Mayo 16, Mayo 18, Mayo 20, Mayo 27, Mayo 30, Mayo 31, Mayo 33, Mayo 36, Mayo 38, Mayo 40, Mayo 41, Mayo 44, Mayo 48, Mayo 54, Mayo 65, Mayo 66, Mayo 82, Mayo 88, Mayo 90, Mayo 93 are normalized to RP_4. Mayo clinic samples: Mayo 03, Mayo 08, Mayo 12, Mayo 14, Mayo 17, Mayo 21, Mayo 23, Mayo 24, Mayo 25, Mayo 26, Mayo 32, Mayo 35, Mayo 43, Mayo 45, Mayo 52, Mayo 55, Mayo 57, Mayo 62, Mayo 63, Mayo 64, Mayo 71, Mayo 73, Mayo 84, Mayo 86, Mayo 89, Mayo 92 are normalized to RP_5. All remaining Mayo clinic samples are normalized to RP_6. ddCT values of target gene signatures are median-centered and scaled to standard deviation of 1 within each of the sample sources (California Pacific Medical Center, Guy's Hospital, Mayo Clinic).
