Perfecta fastmix 2

RNA was extracted from samples to be analysed using an RNAeasy^® Kit (Qiagen, Hilden Germany) and cDNA was synthesised using qScript^® cDNA SuperMix (Quanta Biosciences, VWR, Beverly, MA, USA) according to the manufacturer’s instructions. RT-qPCR was performed with PerfeCTa^® FastMix II (Quanta Biosciences, VWR), using the commercially available FAM-labelled TaqMan Gene Expression Assay for CTEN (Hs00262662_m1; Applied Biosystems, CA, USA) and using the comparative CT method, normalised to human ACTB endogenous control (4326315E, Thermofisher, MA, USA).

For the WST‐1 proliferation assay, 1 × 10⁴ cells were cultured in a 96‐well plate for 24 h in 100 μl of complete media. Then, 10 μl of WST‐1 reagent (Roche) was added to each well. Cells were incubated at 37°C, 5% CO₂ for 4 h, and placed on a shaker for 1 min. The plates were then read on a microplate reader with a wavelength of 420 nm and a reference at 620 nm. For the TaqMan gene expression assay, cDNA was synthesized from 50 ng of glioma cell line‐derived RNA with qScript™ XLT cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). 2 μl of converted cDNA was then used in the qRT–PCR reaction with PerfeCta^® FastMix^® II (Quanta Biosciences), according to the manufacturer's protocol. A custom TaqMan assay specific to wild‐type, but not mutant, PDGFRA was designed by Life Technologies. The fluorescent probe was targeted to the region deleted in PDGFRAΔ⁷, but present in wild‐type PDGFRA. Real‐time PCR and data analysis were performed using the StepOnePlus Real‐Time PCR system (Life Technologies, Carlsbad, CA). GAPDH (Assay ID: Hs02758991_g1) expression was used as the housekeeping gene and relative expression was determined using the 2ΔΔCT method.

Gene expression was performed after extraction of total RNA from PDXs using RNA miniprep system, ReliaPrep^TM FFPE total (Promega) following manufacturer recommendations. cDNA was obtained from 1 μg of total RNA using random primers and M-MLV reverse transcriptase (Amresco) as manufacturer recommendations. GSDMB, HER2, and GAPDH gene expression levels were measured by quantitative real time RT-PCR (qRT-PCR) using pre-designed TaqMan probes (Thermofisher) and PerfeCTa FastmixII (Quanta BioSciences, Inc), on an iQ5 iCycler Realtime PCR Detection System (BioRad), according to the manufacturer's recommendations. All qRT-PCRs were performed in triplicate. Relative GSDMB and HER2 expression was normalized to GAPDH.

Total RNA from cells were isolated with Trizol reagent (Invitrogen, USA) or RNAeasy kit (74106, Qiagen), and 1 μg of RNA was used for reverse-transcription to cDNA using qScript cDNA Supermix (101414–106, Quanta Bio-Sciences) according to manufacturer’s instructions. Real-time RT-PCR was performed using PerfeCTa FastMix II (97065–990, Quanta Bio-Sciences). Fluorescence real-time PCR reactions were run using C100 Touch Thermal Cycler (Bio-Rad) instrument. All primers and probes for quantitation of mRNA for target genes were purchased from IDT or ABI (Applied Biosystems) and FAM-Labeled TaqMan Probe, were normalized to endogenous control eukaryotic 18S ribosomal RNA. (4319413E, Life Technologies)