Abi 7500 rt qpcr system

RNA was extracted from MC‐MSC, WJ‐MSC and BM‐MSC before and after exposure to IFN‐γ using the RNeasy Mini kit (Qiagen, Sussex, UK), following the manufacturer's instructions. RNA was eluted from the spin column with RNAse free water and stored at −80 °C until RT‐qPCR analysis for IDO was performed using the SYBR green mastermix (Applied Biosystems, Warrington, UK) with GAPDH as a reference gene (Qiagen, QuantiTect Primer Assay). The reaction was conducted in the ABI 7500 RT‐qPCR system (Applied Biosystems) at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s then 60 °C for 1 min and data were captured using the sds software (Applied Biosystems). The presence of the IDO mRNA in IFN‐γ‐stimulated cells was expressed as a ratio compared to unstimulated cells, using the comparative threshold method 16. A twofold change threshold (up‐ or down‐regulated) was deemed biologically significant. IDO gene expression was measured over a time course of exposure to IFN‐γ between 1 and 48 h, with the same time point representing the control in normal medium without the addition of the inflammatory cytokine. IDO mRNA levels were initially normalized to the reference gene before calculating the ratio of mRNA in the stimulated versus unstimulated cells.

One microgram of purified RNA was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The reactions were run with the following thermal conditions: 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min. RT-qPCR reactions were carried out to assess the expression levels of CDH1 (Life Technologies, Carlsbad, CA, USA) and EZH2 (Integrated DNA Technologies, Coralville, IA, USA). β2 microglobulin (B2M, Life Technologies) was used as the endogenous control for normalization. The reactions were run in duplicate on 40 ng of cDNA on ABI 7500 RT-qPCR system (Applied Biosystems, Foster City, CA, USA) under the following thermal conditions: 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Expression levels of the target genes were obtained by normalizing the results using the endogenous control B2M. Relative expression was quantified using the comparative 2^−ΔCt method and FC values in gene expression were calculated using the 2^−ΔΔCt method [52 (link),53 (link)]. An FC ≤0.50 and ≥1.5 were used as cut off for downregulation and upregulation, respectively.

Trizol^® reagent (Invitrogen, MA, USA) was used to extract total RNA from cell pellets as per the manufacturer's protocol. For cDNA synthesis, 2 µg of total RNA was converted to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). EBF1, β-actin, IL-6, COX-2, MMP-9, Oct3/4, CAT, SOD2 mRNA expression levels were analyzed with TaqMan gene expression assay using TaqMan probes (EBF1, Hs00395513_m1; β-actin, Hs99999903_m1; IL-6, Hs00174131_m1; COX-2, Hs00153133_m1; MMP-9, Hs00957562_m1; Oct3/4, Hs04260367_gH; CAT, Hs00156308_m1 and SOD2, Hs01553554_m1) on ABI-7500 RT-qPCR system (Applied Biosystems, CA, USA). Relative mRNA expression (fold change) was analyzed with a cycle threshold (Ct) in a linear range of amplification with β-actin as an internal control.