Quantstudio 5 pcr system

One microgram of total RNA from MDA-MB-231 cell was reverse transcribed with oligo dT and M-MLV RT reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed using a GENETBIO SYBR Green Prime Q-master Mix and the QuantStudio 5 PCR system (ThermoFisher). All runs were accompanied by the internal control B2M or HPRT gene. Because both the reference genes yielded very similar results, only B2M results are shown in Figure 6. The samples were run in duplicate and normalized to B2M or GAPDH using a DD cycle threshold-based algorithm, to provide arbitrary units representing relative expression.

Splenic tissue from rats (~30 mg) was stored in RNA later RNA Stabilization Reagent (Qiagen) for 24 hours at 4°C, and thereafter stored at -80°C until extraction. Total RNA extraction was performed using RNEasy Mini kit (Qiagen) and a FastPrep-24 Homogenizer. The kit included an on-column DNA digestion step. cDNA was synthesized from 1 μg RNA using SuperScript III First Strand Synthesis Supermix kit (ThermoFisher Scientific). qPCR was performed using SYBR Green Master Mix (ThermoFisher Scientific) and a Quant Studio 5 PCR system. Primer sequences used are listed in S3 Table. In all experiments Gapdh was used as a reference gene.

Real-time PCR was carried out in 96-well plates using BioMaster HS-qPCR SYBR (2х) (BIOLABMIX LLC, Novosibirsk, Russia) according to the manufacturer's protocol on a QuantStudio5 PCR system (Thermo Fisher Scientific, Waltham, USA). Real-time qPCR analysis of each sample was performed in three replicates. Individual results are provided for each mouse. The relative expression level was determined using the 2^–ΔΔСt method. Tumor-bearing mice not subjected to any treatment (or intact peritoneal macrophages) were used as the control group; the expression level of the target gene in them was assumed to be equal to 1. The GAPDH gene was used as reference. The cycling parameters were as follows: 95°C for 10 min, 40 cycles of 95°C for 30 s, 56°C (Arg1, Ido1, GAPDH)/59°C (IL-1β, TNFα, IL-12a, IL-10, TGF-β1, GAPDH)/64°C (NOS2, GAPDH) for 30 s, 72°C for 30 s, with a final melting step with slow heating from 6 to 95°C.

For assessment of gene expression in cell lines and patient samples, total RNA was reverse transcribed to cDNA using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) or random hexamers (Invitrogen) and Bioscript enzyme (Bioline), respectively. qPCR was performed using a TaqMan® assay kit (Thermo Fisher Scientific) and 18S was used as an endogenous control for data normalization. qPCR was performed using an ABI Prism 7900HT real-time thermal cycler or QuantStudio 5 PCR system (Thermo Fisher Scientific). qPCR data were analysed by the 2^-ΔΔCt (Livak) method [58 (link)].

Total RNA was isolated from the epithelial cells grown in chip using PureLink RNA Mini kit (Thermo Fisher Scientific) and reverse transcribed to cDNA using SuperScript IV Synthesis System (Thermo Fisher Scientific). qRT-PCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) and TaqMan Gene Expression Assays (see Supplementary file 2, Thermo Fisher Scientific) in QuantStudio 5 PCR System (Thermo Fisher Scientific). Relative expression of gene was calculated using 2-∆∆Ct method.

Mouse embryonic stem cells (mESC) were differentiated into dopamine neurons as previously described [79 (link)]. Briefly, mESCs were first differentiated into epiblast stem cells (EPI) using fibronectin-coated plates and N2B27 basal media (composed of Neurobasal media, DMEM/F12, B27 and N2 supplements, l-glutamine and 2-mercaptoethanol) supplemented with FGF2 (10 mg/ml) and activin A (25 mg/ml). After three passages, EPI were differentiated into dopaminergic neurons using plates coated with poly-l-lysine (0.01%) and laminin (10 ng/ml) and N2B7 media supplemented with PD0325901 (1 mM) for 48 h (day 0 to day 2). Three days later (day 5), N2B27 media were supplemented with Shh agonist SAG (100 nM) and Fgf8 (100 ng/ml) for 4 days. The media were then changed to N2B27 media supplemented with BDNF (10 ng/ml), GDNF (10 ng/ml) and ascorbic acid (200 nM) from day 9 onwards. During neuronal differentiation, cells were passaged at day 3 and day 9. Cells were collected for qRT-PCR analysis at several stages: mESC, EPI, day 9 neurons and day 19 neurons. RNA extraction was performed using the RNeasy Mini Kit (Qiagen), and samples were analysed with a QuantStudio 5 PCR system (Thermo Fisher Scientific).