Itaq sybr green pcr master mix

Total RNA was isolated using Trizol RNA extraction method (Invitrogen, CA). cDNA was generated using the SuperScript III reverse transcriptase (Invitrogen) and a mixture of poly-dT and random hexamer primers. Quantitative RT-PCR was performed using gene-specific primers (Additional file 1: Table S1) and iTaq SYBR Green PCR master mix (Biorad, CA). Data was analyzed by standard ΔCq method (2^-ΔΔCq) where ΔCq is the difference between the gene of interest and GAPDH control Cq value. Similar experimental design was used for GBM astrocytes grown in the presence of serum or serum starved for 48 h.

qRT‐PCR assay was used to analyze gene expression profile of NB spheroids. Three groups, including the NB spheroids suspended in culture media (control), bioprinted gelMA containing NB spheroids (NB‐only), and gelMA constructs containing NB spheroid‐HUVEC coculture were cultured in static condition. NB spheroids, either in media or in the gelMA models, were carefully removed via a pipette for RNA extraction using the Aurum Total RNA Mini Kit (Bio‐Rad Laboratories). RNA was reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). qPCR was performed on an Applied Biosystems 7500 real time PCR system (Thermo Fisher Scientific) using the iTaq SYBR Green PCR master mix (Bio‐Rad Laboratories). The relative gene expression was calculated using the 2^−ΔΔCt method with normalization to the Ct of the housekeeping gene glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Multiple NB‐specific primer sequences (Integrated DNA Technologies) were used (Table S1, Supporting Information). Gene expression was assessed at day 7, with each group having three replicates (n = 3 per experimental group).

To extract RNA from liver samples, we used PureLink^® RNA Mini Kit, following the manufacturer’s instructions (Ambion^TM, Invitrogen, Carlsbad, CA, USA). Briefly, lysis buffer, supplemented with 1% β-mercaptoethanol, was added to a liver piece and homogenized. The centrifuged supernatant was transferred to a new tube and the same volume of ethanol 70% was added. After mixing, the whole volume was transferred to the mini-columns. The extracted RNA was eluted with RNAse-free water from the column to a proper tube. The samples were stored at −80 °C, prior to conversion to cDNA and qPCR analysis. Total RNA was transcribed into cDNA by using an NZY First-Strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal). For the amplification of each gene of interest, a corresponding specific pair of primers (STAB Vida, Lisbon, Portugal) was used (Table 2). All reactions were performed in a 20 μL total volume with iTaq SYBR green PCR master mix (Bio-Rad, Hercules, CA, USA). Baseline thresholds were obtained by using the Bio-Rad iQ5 program, and the threshold cycles (CTs) were calculated by the 2CT method, where CT values for the genes of interest were normalized to the level of the hypoxanthine-guanine phosphoribosyl transferase housekeeping gene (Hprt1). Data are shown as n-fold differences relative to values for noninfected samples of Fth1^+/+ genotype, calculated with the 2^−ΔΔCT method.