7500 fast real time pcr machine

RNA was extracted from cancer cells by RNeasy Plus Mini Kit (#74134, Qiagen, Hilden, Germany). RT-PCR was performed using AccessQuick^TM RT-PCR system (#A1700, Promega, Madison, WI, USA) and samples were run on 1.2% agarose gel. The human MUC1 primers used were Forward TGC ATC AGG CTC AGC TTC A, Reverse GAA ATG GCA CAT CAC TCA G, and Tm 60 °C. qPCR primers were designed using the NCBI primer design tool and synthesized by MWG Eurofins (Louisville, KY, USA). The primer sequences will be available upon request. The relative expression levels of multiple genes such as MUC1, PD1, PDL1, LIF, VEGF, IDO1/2, COX1/2, ADAR1, TGFB, TGFBRI/II, PDGF, M6PR, Gal-9, and TRAIL were quantified in cancer cells before and after exposure to mock and CAR T cells using Applied Biosystems^® 7500 fast Real-Time PCR machine and SYBR Green PowerUp Master Mix (A25742, Life Technologies). Relative expression of each gene was calculated as 1/(gene CT – GAPDH CT).

Cells grown in 12-well plates were transfected with siRNAs for 48 h. Total RNA was extracted using an Invisorb Spin Cell RNA mini kit (Invitek Molecular, Berlin, Germany) according to the manufacturer’s protocol, and RNA concentration was determined using a NanoDrop3000 (Thermo Scientific). 500 ng of total RNA was used to perform cDNA synthesis using a High Capacity cDNA Reverse Transcription kit (Life Technologies) according to the manufacturer’s protocol. Real-time quantitative PCR was performed using Fast SYBR Green PCR MasterMix (Life Technologies) in a 7500 Fast real-time PCR machine (Life Technologies). One-twentieth of the cDNA was used as a template for the qPCR reaction, and 200 nM of each primer was used (see Supplementary Table S2). Three biological replicates were performed. The results were acquired using the -ΔCt method, with mRNA levels from siRNA-treated cells being normalised to those found in cells treated with non-silencing (NEG) siRNAs.

For the analysis of gene expression, eNSCs were seeded into 6-well plates and differentiated according to the experimental protocol (see above) in six independent experiments. At the end of the differentiation process (DIV 18), the medium was collected for further proteomic analysis and cells were harvested by adding 700 μL of RNA Lysis Buffer (Zymo Research, Irvine, CA, USA) and stored at −80 °C until further processing. RNA was isolated using a Quick-RNA^TM MiniPrep (Zymo Research, Irvine, CA) according to the manufacturer’s instructions and its quality was assessed by agarose gel electrophoresis. cDNA was synthesized from 500 ng of total RNA in a 20 μL reaction volume using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Waltham, MA) according to the manufacturer’s protocol. The quality of cDNA and possible genomic DNA contamination was controlled by amplification of the reference gene Hprt1 by qPCR. Expression of target and reference genes was assessed with TaqMan^® assays (probe information is listed in Supplementary Table S1) and TaqMan^® Gene Expression Master Mix using a 7500 Fast Real-Time PCR machine (Life Technologies, Carlsbad, CA, USA) with normalization to Gapdh, Hprt1 and Actb expression. Ct values were obtained by 7500 software (ABI) and the threshold was set to 0.3 for all genes and experiments.