Gene Expression Analysis of DK210 (EGFR) treatment study in a syngeneic tumor model
Description
Transcriptional gene analysis from tumor infiltrating effector memory CD8 T cells in a DK210 (EGFR) study with B16F10 tumor bearing mice
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Growth protocol: Mice were maintained under climate controlled, specific pathogen-free conditions in the animal facilities of Deka Biosciences. B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; ATCC or Quality Biological) with 10% fetal bovine serum (FBS; GeminiBio) and 1 ug/mL Puromycin (InvivoGen). Treatment protocol: C57BL/6 mice were implanted subcutaneously with a human EGFR expressing B16F10 cell line (B16F10hEGFR+). B16F10hEGFR+ tumor bearing mice were treated 3 times per week with vehicle control or DK210 (EGFR) (2 mg/kg). Cell lysates were prepared by re-suspending sorted effector memory CD8+ TILs in 350 µl Buffer RLT provided in the RNeasyTM Mini Kit (Qiagen), vortexing for 1 minute, and freezing at -80°C prior to RNA extraction and nCounter® analysis Extract protocol: Total RNA from sorted cells was extracted using the Qiagen RNeasyTM Mini Kit according to manufacturer recommendations. The purity and concentration of RNA was measured using a NanoDrop One (Thermo Scientific). Label protocol: See manufacturer's website,NanoString Technologies hyb protocol: RNA from each sample was hybridized using the PanCancer IO 360™ Panel of 770 genes. scan protocol: Gene expression analysis using a nCounter® Pro Analysis System was performed by Bruker (Seattle, WA). data processing: Absolute read counts were extracted using the nSolver® Analysis Software (version 4.0). value definition: Target genes were normalized to 12 housekeeping genes (Abcf1, Dnajc14, Ercc3, G6pdx, Gusb, Oaz1, Polr2a, Psmc4, Pum1, Sdha, Tbc1d10b, Tmub2). GEO ncounter Platform: GPL34315