Quantstudio 5 rt qpcr system

RNA extractions from hMDMs and hAMs were performed using an RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. RNA content and quality was quantified and assayed, respectively, using a Nanodrop (Thermo Fisher Scientific) and RNA reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (VWR). Catalogued pre-designed gene primer probes for IL1β, TNFα, IL10, NFκB, IL12a, IL18, PFKFB3, GAPDH, PKM2, G6PD, RPIA, CPT1A, FASN, GLS, IDO1, and 18S were purchased and real-time RT-qPCR was performed using Taqman Universal Master Mix (Applied Biosystems) on a QuantStudio 5 RT-qPCR System (Applied Biosystems). Relative quantitative data was obtained and analyzed utilizing the 2^–ΔΔCt method as previously described (27 (link)). Secreted protein levels of IL1β, IL10 (BioLegend ELISA Max Deluxe kits) and TNFα (Invitrogen ready-set-go kit) present in cell supernatants were quantified by ELISA, according to the manufacturer’s instructions.

Total RNA was extracted from cells using TRIzol (Invitrogen) (16 (link)). cDNA synthesis was performed by PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). RT-qPCR was performed using primers (Table 2) and TB Green™Premix Ex Taq™II (Takara) in a QuantStudio™ 5 RT-qPCR System (Applied Biosystems). All of operations above were followed according to the manufacturer's instructions. Expression levels were determined by a standard ΔΔCt method. β-Actin was analyzed as an internal control.

RNA extractions from monocytes were performed using an RNeasy Mini Kit (Qiagen, catalog 74136) according to the manufacturer’s instructions. RNA content and quality were quantified and assayed, respectively, using a Nanodrop (Thermo Fisher Scientific) and RNA reverse transcribed using the SensiFast Reverse-Transcription Kit (Meridian Biosciences, catalog BIO-65054). Cataloged TaqMan (Thermo Fisher Scientific) predesigned gene primer probes attached to the FAM dye were used: GPI (Hs00976715_m1), PFKFB3 (Hs00998698_m1), GAPDH (Hs02786624_g1), PKM2 (Hs00761782_s1), and ATP5B (Hs00969569_m1). 18S (Hs03003631_g1) was used as the reference gene primer, attached to the VIC dye. RT-qPCR was performed using SensiFast Probe Mix (Meridian Biosciences, catalog BIO-82005) on a QuantStudio 5 RT-qPCR System (Applied Biosystems). Relative quantitative data were obtained and analyzed utilizing the 2^–ΔΔCt method.