Rneasy ffpe rna kit

The 21-gene RS assay was performed locally at our centre. Detailed information on the 21-gene RS analysis was presented in our previous work.^{15 (link)} In brief, macrodissection was performed to ensure that tumour elements accounted for more than 50% of the tissue. Subsequently, RNA was extracted from three 10-μm unstained sections of formalin-fixed, paraffin-embedded (FFPE) tissue using the RNeasy FFPE RNA kit (Qiagen, 73504, Germany). Gene-specific reverse transcription was performed using an Omniscript RT kit (Qiagen, 205111, Germany). Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done using Premix Ex Taq^TM (TaKaRa Bio, RR390A) in an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA). In this assay, the expression of 16 cancer genes is measured in triplicate and normalised to a set of 5 reference genes. The RS result, ranging from 0 to 100, was derived from the reference-normalised expression of the 16 cancer genes. According to the 21-gene RS results, patients were categorised into low-risk (RS < 18), intermediate-risk (RS 18–30) and high-risk (RS ≥ 31) groups.^{9 (link),10 (link),12 (link)} For further analysis, the individual gene expression of the 16 cancer genes was measured, and the distribution of the 16-cancer gene expression in IDC and IDC/DCIS patients was analysed.

The 21-gene RS testing was performed on formalin-fixed, paraffin-embedded tissue sections as we previously described. In brief, hematoxylin and eosin-stained slides were used to identify enough tumor tissues. Then, total RNA was extracted by the RNeasy FFPE RNA kit (Qiagen GmbH) from two 10-μm unstained sections after verifying the absence of DNA contamination. Gene-specific reverse transcription was performed using the Omniscript RT kit (Qiagen GmbH) and standardized quantitative RT-PCR was accomplished in 96-well plates using an Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.). The expression levels of each gene were measured in triplicate, and expression levels of cancer-associated genes were normalized by 5 reference genes. Finally, the RS was calculated with a specific algorithm as previously described and categorized as low-risk (RS ≤ 25) and high-risk (> 25).

The detailed information of 21-gene RS evaluation was described in our previous work^{19 (link)}. RNA was extracted from three 10μm unstained sections of FFPE breast tumor tissue, which was prepared by experienced pathologists in the Department of Pathology, using RNeasy FFPE RNA kit (Qiagen, 73504, Germany). Reverse transcription was performed using Omniscript RT kit (Qiagen, 205111, Germany). Quantitative RT-PCR was accomplished using Premix Ex TaqTM (TaKaRa Bio, RR390A) in Applied Biosystems 7500 Real-Time PCR System (Foster City, CA). Gene expression was verified in triplicate, and normalized to five endogenous reference genes. Gene-specific normalized cycle threshold value was applied to calculate RS. For patients with multifocal disease, the highest RS was recorded.