A validated single-cell-based strategy to identify diagnostic and therapeutic targets in complex diseases [breast cancer validation cohort]

Breast cancer validation study of CD4+ T cells in an independent group of 24 patients (median age [range], 61.5 [35–88] years) was recruited, based on the same inclusion criteria. All recruited patients were women. Additionally, 14 independent healthy controls were profiled. Total RNA was extracted using the AllPrep DNA/RNA Micro kit (Qiagen, Hilden, Germany; cat. no. 80284) according to the manufacturer’s instructions. RNA concentration and integrity were evaluated using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, California, USA; cat. no. 5067-1511) on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Microarrays were then further computationally processed as described in One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling protocol (Agilent Technologies, Santa Clara, California, USA).