Qpcr master mix

RNA was extracted from SW872 adipocytes using an RNeasy kit (Qiagen). Between 400 and 1000 ng of RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). qPCR was performed with an Applied Biosystems ABI-PRISM 7900HT Sequence Detection System. Gene expression was normalised to TATA binding protein (TBP) using Assays on Demand and QPCR master mix (Applied Biosystems). The following TaqMan^® Gene Expression Assays (Applied Biosystems) were used: TBP (Hs00427620_m1) and CXCL10 (Hs01124251_g1).

Total RNA was isolated from BMMCs using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). RNA was quantified on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using the ReverTra Ace RT-PCR Kit (TOYOBO, Osaka, Japan). Quantitative real-time PCR (qPCR) was performed on a Step One Plus Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using qPCR Master Mix (Applied Biosystems) with specific primers and probes against mouse REV-ERB-α, IL-13 (Applied Biosystems), REV-ERB-β, IL-6, and GAPDH (IDT; Coralville, IA, USA). qPCR data were normalized against the corresponding levels of GAPDH mRNA.

Reactions were performed in triplicate, using 2ul of template per reaction of cDNA in qPCR master mix, (Applied Biosystems). The Quantitative- Comparative C_T (ΔΔC_T) program on the machine was used for the qPCR. The following taqman primers/probes (ThermoFisher Scientific, Applied Biosystems) were used: ADAM23 (Hs00187022_m1), NRCAM (Hs01031598_m1), CNTNAP2 (Hs01034296_m1), LTBP1 (Hs01558763_m1), ITGB5 (Hs00174435_m1), TAL1 (Hs01097987_m1), CLU (Hs00156548_m1), ALOx12 (Hs00167524_m1), and ADRA2A (Hs01099503_s1). A GAPDH (Hs02786624_g1) internal control assay was used. All the Taqman expression assays had a FAM dye except the GAPDH, which had a VIC dye. Both the internal control gene (GAPDH) and the gene of interest were ran in the same reaction.

RNA was extracted from HAECs, HUVECs or HEK-293 cells using an RNeasy kit (Qiagen). Between 400–1000 ng of RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). qPCR was performed with an Applied Biosystems ABI-PRISM 7900HT Sequence Detection System. Gene expression was normalized to TATA binding protein (TBP) using QPCR master mix (Applied Biosystems) and the following TaqMan® Gene Expression Assays (Applied Biosystems): TBP (Hs00427620_m1), SLC5A1 (SGLT1, Hs01573793_m1), SLC5A2 (SGLT2, Hs00894642_m1), IL6 (Hs00174131_m1) and CCL2 (MCP-1, Hs00234140_m1).

RNA was extracted from cells 48 h after infection using an RNeasy Plus minikit (Qiagen) according to the manufacturer's protocol. cDNA was prepared using a High-Capacity cDNA reverse transcription kit (Thermo Fisher) and cleaned up using a DNA purification kit (Qiagen). Water-only and no-RT controls were included in the experiments as negative controls. Quantitative PCR was carried out with qPCR Mastermix (Applied Biosystems) following the manufacturer’s instructions on a StepOnePlus real-time PCR system (Applied Biosystems) using the following cycling parameters: 1 cycle at 95°C for 8 min and 45 cycles at 95°C for 10 s followed by 60°C for 1 min. The primers and probes (Sigma) used were as follows: Tat1 forward, 5′-AGA TCT CTC GAC GCA GGA CT-3′; Tat1 reverse, 5′-GGC TGA CTT CCT GGA TGC TT-3′; D1A3 probe (tat1), 5′-6-carboxyfluorescein (FAM)-TCG ACA CCC AAT TCA GTC GC-6-carboxytetramethylrhodamine (TAMRA)-3′; Tat2 forward, 5′-GGA CAG CAG AGA TCC AGT TTG-3′; Tat2 reverse, 5′-GAT GCT TCC AGG GCT CTA GTC-3′; D2A3 probe (tat2), 5′-FAM-GTC GAC ACC CAA TTC TTT CCA G-TAMRA-3′; All-tat/vpr forward, 5′-TCC TAT GGC AGG AAG AAG CG-3′; All-tat/vpr reverse, 5′-AGC TTG ATG AGT CTG ACT GT-3′; All-tat/vpr probe, 5′-FAM-TCT GAT GAG CTC TTC GTC GCT GTC TC-TAMRA-3′.