Rt2 real time sybr green rox master mix

Total RNA (1.5 μg) was used for the synthesis of cDNA using the ThermoScript RT-PCR system for first strand synthesis (Invitrogen – Life Technologies, Grand Island, NY, USA). QPCR reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmol of the primers in a final volume of 25 μl of 2 × concentrated RT2 Real-Time SYBR Green/ROX master mix (Qiagen) in an Applied Biosystems 7300 Real-Time PCR instrument (Norwalk, CT, USA). The cycle parameters were: 50°C for 2 minutes, one denaturation step at 95°C for 10 minutes and 40 cycles of denaturation at 95°C for 10s followed by annealing and elongation at 60°C. Relative gene expression of each transcript was normalized to GAPDH using the ΔΔCt method. Primer sequences for GAPDH were: forward: 5′-CTGCACCACCAACTGCTTAG-3′, reverse: 5-GGGCCATCCACAGTCTTCT-3, and for LPA₂: forward 5-CCAGCCTGCTTGTCTTCCTA-3, and reverse: 5-GTGTCCAGCACACCACAAAT-3. For ATX gene product QPCR measurements 0.1 × 10⁶ IEC-6 cells were plated per well of a 6-well plate in complete growth media. The following day, cells were serum-starved for 24 hours prior to stimulation with 10ng/ml of rat TNFα (R&D Systems) for 15 min, 3 h and 6h. The ATX forward primer was 5′-ATTACAGCCACCAAGCAAGG-3′ and the reverse 5′-GGCAGAGAAAGCCACTGAAG -3′.

Total RNA (1.5 μg) was used to generate cDNAs using the ThermoScript RT-PCR system to synthesize the first strand (Invitrogen). Quantitative PCR (qPCR) reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmoles of primers in a final volume of 25 μl of 2× concentrated RT2 Real-Time SYBR Green/ROX master mix (Qiagen) in an Applied Biosystems QuatStudio 6-Flex Real-Time PCR instrument (Norwalk, CT, USA). The cycle parameters were: 50 °C for 2 min, one denaturation step at 95 °C for 10 min and 40 cycles of denaturation at 95 °C for 10s followed by annealing and elongation at 60 °C. The relative gene expression of each transcript was normalized to GAPDH using the ΔΔCt method. Sequences of primers used for qPCR are provided in Table S1.

Total RNA (1.5 μg) from cerebral cortex samples was used to generate cDNAs using the ThermoScript RT-PCR system for first-strand synthesis (Invitrogen). Quantitative PCR (qPCR) reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmoles of primers in a final volume of 25 μl of 2× concentrated RT2 Real-Time SYBR Green/ROX master mix (Qiagen) in an Applied Biosystems QuantStudio 6 Flex Real-Time PCR instrument (Norwalk, CT, USA). The cycle parameters were: 50°C for 2min, one denaturation step at 95 °C for 10 min and 40 cycles of denaturation at 95°C for 10 seconds followed by annealing and elongation at 60°C. The relative gene expression of each transcript was normalized to GAPDH using the ΔΔCt method. Primer sequences for TLR-4, IL-1β, IL-6, TNF-α, and MCP-1 were chosen according to the previous publication [15 ].

Analysis of specific mRNA by RT-qPCR was performed as described before (Shukla et al., 2018 (link)). Total RNA (1.5 μg) was used to generate cDNAs using the ThermoScript RT-PCR system for first-strand synthesis (Invitrogen). Quantitative PCR (qPCR) reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmol of primers, in a final volume of 25 μL of 2× concentrated RT2 Real-Time SYBR Green/R.O.X. master mix (Qiagen, Germantown, MD), using an Applied Biosystems QuatStudio 6-Flex Real-Time PCR instrument (Norwalk, CT, USA). The cycle parameters were 50°C for 2 min, one denaturation step at 95°C for 10 min, 40 cycles of denaturation at 95°C for 10s, followed by annealing and elongation at 60°C. The relative gene expression of each transcript was normalized to GAPDH using the ΔΔCt method. Sequences of primers used for qPCR are provided in Table S1.