Applied biosystem 7500 real time pcr detection system

Determination of IL-6, TNF-α, and TGF-B genes expression by real-time PCR (RT-qPCR).
RNA purification was performed using TRIzol reagent (Invitrogen, USA). Complementary DNA was then synthesized using Thermo Scientific Maxima First Strand cDNA Synthesis Kit with dsDNase (Thermo Fisher Scientific, Rockford, USA).
Primer sets for genes were designed using the Primer3PLUS software (v. 0.4.0; http://frodo.wi.mit.edu/;Table 1). Real-time PCR assays were performed using the Applied Biosystem 7500 real-time PCR detection system (Life Technologies, USA) according to the method described [20 (link)] with the SensiFAST SYBR Lo-ROX PCR Master Mix Kit (Bioline, United Kingdom). The total reaction volume was 20 μl, and the thermal reaction profile was as follows: initial denaturation at 95°C for 2 min and then 40 cycles of 95°C for 5 s followed by 60°C for 30 s. Calculation of fold change of gene expression was normalized to β–actin according to cycle threshold (Ct) method (2^-ΔΔCt). The relative gene expression for all genes was normalized to one in the control group (negative control) [21 (link)].

The TRIZOL reagent was used to purify the RNA (Invitrogen, Waltham, MA, USA). Thermo Scientific Maxima First Strand cDNA 161 Synthesis Kit with dsDNase was then used to make complementary DNA (Thermo Fisher Scientific, Rockford, IL, USA). The Primer3PLUS program (v. 0.4.0; 163 http://frodo.wi.mit.edu/ (accessed on 25 December 2021); Table 3) was used to create gene primer sets. According to the method given by Freeman et al., real-time PCR tests were performed using the Applied Biosystem 7500 real-time PCR detection system (Life Technologies, Waltham, MA, USA) [70 (link)] with the SensiFAST SYBR Lo-ROX PCR Master Mix Kit (Bioline, London, UK). The entire reaction volume was 20 μL, with the following thermal reaction profile: Denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s, followed by 60 °C for 30 s. The fold induction values were calculated using the Cycle threshold (Ct) method (2^−ΔΔCt) [71 (link)].

TRIzol reagent (Invitrogen, Waltham, MA, USA) was used to purify the RNA. Thermo Scientific Maxima First Strand DNA Synthesis Kit with dsDNase was then used to make complementary DNA (Thermo Fisher Scientific, Rockford, IL, USA). The control gene was the B actin gene. Primer 3 PLUS software was used to create gene primer sets (v. 0.4.0; 163 https://frodo.wi.mit.edu/; accessed on 29 December 2021 Table S1). Real-time PCR assays were conducted using the Applied Biosystem 7500 real-time PCR detection system (Life Technologies, Carlsbad, CA, USA) with the SensiFAST SYBR Lo-ROX PCR Master Mix Kit (Bioline, Toronto, UK). The overall reaction volume was 20 μL, with the following thermal reaction profile: initial denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C for 5 s, then 60 °C for 30 s. The method (2^−ΔΔCt) was used for the calculation of fold induction values [43 (link)].