Qpcr abi 7500 fast real time pcr system

QTotal RNA was isolated using the RNeasy kit (QIAGEN, Germany), and single stranded cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher, USA). The qPCR was performed using TaqMan™ Fast Advanced Master Mix (Thermo Fisher, USA) and a qPCR ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The following primers were used 1) Hs00164383_m1, CYP1B1, FAM-MGB 2) Hs00174128_m1, TNFalpha, FAM-MGB 3) Hs00420895_gH, RPLPO, FAM-MGB 4) Hs01005075_m1, AHRR, FAM-MGB 5) Hs01054794_m1, CYP1A1, FAM-MGB (all Thermo Fisher, USA). Fold changes were calculated by the ΔΔc_t-Methode relative to the unstimulated control with RPLP0 as the reference (52 (link)).

For quantitative real-time PCR (qPCR), RNA isolation and reverse transcription was done as described previously (60 (link)). Briefly, RNA was isolated using the RNeasy Mini kit with DNase I digestion (both Qiagen, Germany). 500ng RNA was used to synthesize cDNA using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, USA). The qPCR was performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems, USA) on a qPCR ABI 7500 FAST Real-Time PCR System (Applied Biosystems, USA) using TaqMan™ primer sets for human TWIST1, MTHFD2 and SDHA (Applied Biosystems, USA). Gene expression was measured by the ΔΔ_ct-Method relative to the unstimulated control and with SDHA as references. Levels of significance were determined by Student’s t-test in GraphPad Prism 9.

The cells were treated for 6 h with ligands with or without LPS. Consecutively, the total RNA was isolated using the RNeasy kit (QIAGEN, Hilden, Germany). Single-stranded cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The qPCR was performed with a qPCR ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) under usage of TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The following primers were used: (1) Hs00164383_m1, CYB1B1, FAM-MGB (2) Hs00174128_m1, TNFalpha, FAM-MGB (3) Hs00420895_gH, RPLPO, and FAM-MGB (all Thermo Fisher, USA). Fold changes were calculated by the ΔΔct-method relative to the unstimulated control with RPLP0 as the reference [29 (link)].