Formalin-fixed paraffin-embedded material was cut in five 10μm-thick pieces per sample. Extraction of RNA was done according to the manufacturer’s instructions (ExpressArt FFPE Clear RNAready Kit, AmpTec, Hamburg, Germany). Fresh-frozen blocks’ RNA was extracted as described in Hummel et al, NEJM 2006). The RNA quality was analyzed with the Agilent RNA 6000 Nano Chips (Agilent Technologies, Santa Clara, California, USA) following the product’s protocol. Further material quality control is present after RNA processing at the nCounter instrument for digital multiplexed gene expression due to the company’s recommendations.
Label
NA
Label protocol
NA
Hybridization protocol
Straight after a RNA quality measurement and with a known RNA concentration for each sample, 300ng were able to be hybridised with our custom-designed Reporter Probe (each with a specific 5’-end colour code signal) and Capture Probe (marked with biotin at the 3’ end). !Sample_scan_protocol = After overnight hybridisation the samples were ready to load to the PrepStation where they were washed with a two-step magnetic bead-purification process and loaded in the 12-well cartridges. Afterwards the cartridges were loaded to the Digital Analyzer (DA, nCounter, NanoString Technologies Inc., Seattle, WA, USA). The DA resolution was set to high, meaning that 280 fields of view (FOV) were counted per sample. A comma-separated-value (.csv) file was obtained after processing.
Scan protocol
Straight after a RNA quality measurement and with a known RNA concentration for each sample, 300ng were able to be hybridised with our custom-designed Reporter Probe (each with a specific 5’-end colour code signal) and Capture Probe (marked with biotin at the 3’ end). After overnight hybridisation the samples were ready to load to the PrepStation where they were washed with a two-step magnetic bead-purification process and loaded in the 12-well cartridges. Afterwards the cartridges were loaded to the Digital Analyzer (DA, nCounter, NanoString Technologies Inc., Seattle, WA, USA). The DA resolution was set to high, meaning that 280 fields of view (FOV) were counted per sample. A comma-separated-value (.csv) file was obtained after processing.
Data processing
Data processing was done using the R-package NanoStringNorm (Waggot et al, Bioinformatics 2012). Final data normalization was performed using the quantile normalization (Bolstadt et al, Bioinformatics 2003)
Molecular classification of mature aggressive B cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens [Frozen tissue]
Molecular classification of mature aggressive B cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens
