Total RNA was isolated from fresh-frozen ccRCC and metastasis tissue using the mirVana™ miRNA Isolation Kit (Life Technologies) as previously described [18 (link), 19 (link)]. Genome-wide transcriptome analyses were performed using Human Transcriptome Array HTA 2.0 (Affymetrix) according to the manufacturer’s protocol. Further processing of microarray data were performed as previously described [18 (link)] (Additional file 1 : Supplementary methods). Gene expression data (generated using HuGene 1.0ST Affymetrix array) from 53 sunitinib-treated ccRCC patients were downloaded from ArrayExpress (E-MTAB-3267).
Quantitative real-time PCR (qRT-PCR) was performed using TaqMan technology on a BioMARK System (Fluidigm) as described previously [18 (link), 19 (link)]. TaqMan gene expression assays for 97 genes of the S3-score, as well as five genes used for normalization were purchased from Life Technologies. Further details about calculation of the S3-score based on interprofile correlations and development of a S3-score calculation model for use of qRT-PCR data are provided in the Additional file1 : Supplementary methods.
Quantitative real-time PCR (qRT-PCR) was performed using TaqMan technology on a BioMARK System (Fluidigm) as described previously [18 (link), 19 (link)]. TaqMan gene expression assays for 97 genes of the S3-score, as well as five genes used for normalization were purchased from Life Technologies. Further details about calculation of the S3-score based on interprofile correlations and development of a S3-score calculation model for use of qRT-PCR data are provided in the Additional file
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