Human gapdh

Samples were collected using inclusion and exclusion criteria described above. See Table 2 for demographics of the patients included for RT-PCR. Total RNA was isolated as described above. cDNA was prepared using the High Capacity cDNA Reverse Transcription Kit (Cat#4368814; Thermo Fisher Scientific, Waltham, MA). Quantitative real-time PCR (RT-PCR) was performed as previously described [18 (link)]. Amplification of target sequences was detected by a sequence detector ABI 7500 Fast RTPCR system (Applied Biosystems, Foster City, CA) using TaqMan Fast Universal PCR Master Mix and CXCL14 (Assay ID Hs01557413_m1; Thermo Fisher Scientific, Waltham, MA) or C3 (Assay ID Hs00163811_m1; Thermo Fisher Scientific, Waltham, MA) or ERAP2 (Assay ID Hs01073631_m1; Thermo Fisher Scientific, Waltham, MA) or CHIT1 (Assay ID Hs00185753_m1; Thermo Fisher Scientific, Waltham, MA) specific primers. The reactions were run in duplicates of 25 μl, including the endogenous control, human GAPDH (Assay ID Hs02786624_g1; Thermo Fisher Scientific, Waltham, MA), for each individual gene expression assay. For quantitative analysis, comparative delta-delta C_t was used to normalize the data based on the endogenous reference and to express it as the relative fold change after the exclusion criteria were verified by comparing primer efficiencies.

Total RNA from HepG2 cells, liver tissues, and epididymal WAT was extracted using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany, #74134), and RNeasy Lipid Tissue Mini kit (Qiagen, Hilden, Germany, #74804) respectively according to manufacturer’s instructions followed by reverse transcription (RT) for cDNA synthesis (Thermo Fisher Scientific, #11754050). Real-time-quantitative PCRs (RT qPCRs) were performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using TaqMan Gene Expression Master Mix (Applied Biosystems). Probes (mouse Acbp: #Mm01286585_g1, mouse Pparg: #Mm00440940_m1, mouse Ppia: #Mm02342430_g1, human ACBP: #Hs01554584_m1, human PPARG: #Hs01115513_m1, and human GAPDH: #Hs03929097_g1) were purchased from Thermo Fisher Scientific.

Total RNA was isolated from cells using RNeasy kit (Qiagen). cDNA was obtained using iScript™ cDNA Synthesis Kit (Bio-Rad). Real-time quantitative PCR assays were performed using TaqMan Gene Expression Master Mix and the probes human NEMO/IKBKG (Hs00415849_m1) and human GAPDH (Hs03929097_g1) (ThermoFisher Scientific). Amplifications were run in a Bio-Rad CFX96 real-time PCR system, using the following protocol: 50 °C for 2 min, 95 °C for 10 min, 39 cycles of 95 °C for 15 s, 55 °C for 1 min. Each value was normalized to GAPDH levels. Relative expression levels were determined using the 2^−ΔΔCt method. Real-time PCR was controlled by the Bio-Rad CFX Manager v 3.1 software.

Quantitative RT-qPCR was carried out in a total reaction volume of 10 µL containing 1× each of TaqMan gene expression assays (20×) (human IL10 (Hs00174086_m1), and human IL15 (Hs01003716_m1), ThermoFisher Scientific), 1× TaqMan Fast Advanced Master Mix (2×) (ThermoFisher Scientific), and 10 ng of cDNA. RT-qPCR reactions were performed in a StepOnePlus Real-Time PCR Systems thermocycler (ThermoFisher Scientific), and relative expression of target genes was normalized by human GAPDH (Hs99999905_m1; Thermo Fisher Scientific), a very common reference gene used in the literature and highly standardized in our laboratory [13 (link),23 (link)]. RT-qPCR data analysis was performed with the N₀ method implemented in LinRegPCR v. 2020.0, which considers RT-qPCR mean efficiencies estimated by the window-of-linearity method [28 (link),29 (link)]. Briefly, N₀ values were calculated in LinRegPCR using default parameters. Then, N₀ values from the gene of interest (GOI) were normalized by taking their ratio to the N₀ of the reference gene GAPDH (N_0GOI/N_0REF).

Reverse transcription was performed using qScript cDNA Synthesis Kit (QuantaBio). The mRNA expression was determined by 7900 HT Fast Real time PCR System (Thermo Fisher Scientific). Master mix for real-time PCR was PerfeCTa qPCR FastMix II, ROX (VWR International, Radnor, PA, Cat# 97065-998), and TaqMan probes were used as follows: human GAPDH (Hs02758991_g1, Life Technologies), human PARP14 (Hs00981511_m1), human IL-1β (Hs00174097_m1), human CCL2 (Hs00234140_m1), human CXCL9 (Hs00171065_m1), human CXCL10 (Hs01124252_g1), human CXCL11 (Hs04187682_g1). The expression levels were normalized to human GAPDH (Hs02758991_g1).