Two tissue curls from the FFPE block of each biopsy sample were dissected at 16-μm thickness per curl and stored at -80°C until RNA extraction was performed with either Zymo® Quick-RNA FFPE kits by manual RNA extraction or with Zymo® Quick-RNA MagBead kit and Tecan Fluent Liquid Handler.
Label
N/A
Label protocol
N/A
Hybridization protocol
150 ng total RNA (in 8 μl) was hybridized to gene-specific probes at 65˚C for 16 hours and then deposited onto glass cartridges using the standard sensitivity” setting on the nCounter® Prep Station (NanoString Technologies, Seattle, WA, USA). When total RNA yield of one sample was <150 ng, all obtained total RNA from that sample was used as input for hybridization. Samples with low RNA concentration were vacuum concentrated to reduce the RNA sample volume to 8 μl or less before hybridization.
Scan protocol
The number of barcodes hybridized to specific RNA targets on cartridges were directly counted by the nCounter® Digital Analyzer with high fields of view setting of 280.
Data processing
Normalization of RNA loading was performed using the geometric mean of 12 internal reference genes (housekeeping genes; ABCF1, G6PD, GUSB, NRDE2, OAZ1, POLR2A, PPIA, SDHA, STK11IP, TBC1D10B, TBP, and UBB).
Gene expression data of formalin-fixed-paraffin-embedded (FFPE) renal biopsy tissue assessed with Banff Human Organ Transplant (B-HOT) gene panel assay on NanoString nCounter®
Data table header descriptions
ID_REF
VALUE
Log2 transformed and normalized count data with batch effect correction for the B-HOT gene panel assay
