21-Gene Expression Analysis of FFPE Tissue

The 21-gene tests were performed on formalin-fixed, paraffin-embedded tissue. Hematoxylin and eosin-stained slides were deparaffinized into two 10-µm unstained sections using xylene followed by ethanol as we described in our previous study (15 (link)). RNA was extracted and purified using the RNeasy FFPE kit (QIAGEN, Hilden, Germany). Gene-specific reverse transcription was conducted using Omniscript RT kit (Qiagen, 205111, Germany). Standardized quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in 96-well plates with Applied Biosystems (Foster City, CA, USA) 7500 Real-Time PCR system. RT-PCR was carried out with the Omniscript RT kit (Qiagen, Valencia, CA, USA). Expression of each gene was measured in triplicate, and normalized relative to a set of five reference genes.