Total rna zol out kit

After cell lysis in TRI Reagent (ThermoFisher), Direct-zol RNA MiniPrep kit (ZymoResearch) or Total RNA Zol-Out kit (A&A Biotechnology) was used for total RNA isolation. For Flp-In T-REx-293 cell lines, a fraction of lysates prepared in Cytoplasmic Lysis Buffer [PBS, 0.1% NP40, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)] was mixed with four volumes of TRI Reagent for further isolation. The concentration of isolated total RNA was assessed by measurement at 260 nm using DeNovix spectrophotometer. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random hexamer primers (Promega), according to the manufacturer's protocols. RT-qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and CFX Connect Real-Time System (Bio-Rad), according to the manufacturer's protocols and established guidelines for qPCR. Digital droplet PCRs (ddPCRs) were prepared using DG8 cartridges and gaskets, QX200 Droplet Generation Oil and QX200 EvaGreen Digital PCR Supermix (BioRad) and performed on QX200 Droplet Digital PCR System (BioRad), according to the manufacturer's protocols. All primer sequences are listed in Table S4.

Total RNA was extracted with the Total RNA Zol-Out Kit (A&A Biotechnology) and converted to cDNA using NxGen^TM M-MuLV reverse transcriptase (Lucigen) with a mix of random hexamers (Invitrogen) and oligo (dT) (GenoMed). Real-time PCR was performed on the CFX96 thermocycler (Bio-Rad Laboratories) using SYBR Green I containing a Universal PCR Master Mix (A&A Biotechnology) and primers specific for mouse camp (5′-CTTCAAGGAACAGGGGGTGG-3′, 5′-ACCTTTGCGGAGAAGTCCAG-3′) and two housekeeper genes B2M (5′-GGACTGGTCTTTCTATATCCTGGC-3′, 5′-GA TCACATGTCTCGATCCCAGTAG-3′) and GAPDH (5′-TGT GTCCGTCGTGGATCTGA-3′, 5′-TTGCTGTTGAAGTCGCA GGAG-3′). Gene expression normalized to geometric mean of the housekeeper genes was calculated using the 2^–ΔCt method (Vandesompele et al., 2002 (link); Nessi et al., 2010 (link)).

Total RNA was isolated from the cells using Trizol (Thermo Fisher) and the Total RNA Zol out kit (A&A Biotechnology) and residual genomic DNA digested with DNAseI (Life technologies). cDNA synthesis reactions were performed with Superscript II Reverse Transcriptase (Life Technologies) kit following with random priming and manufacturer's instructions. RT-qPCR reactions were conducted CFX Connect Real-Time PCR Detection System thermocycler (BioRad) using GoTaq qPCR Master Mix (Promega) following manufacturer protocol. Amplification was performed on the using following thermal program starting with 3 min at 95°C, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The specificity of the RT-qPCR products was assessed by melting curve analysis. Primers used for real-time PCR are listed in Supplementary Table S5. Expression data was calculated using 2-ΔΔCt method (38 (link)). Cyclophilin A (CyPA), GAPDH and β2 microglobulin (B2M) were used as housekeeping genes for the analysis.

Total RNA was extracted with the Total RNA Zol-Out Kit (A&A Biotechnology, Gdynia, Poland) and converted to complementary DNA using NxGen M-MulV reverse transcriptase (Lucigen Corporation, Middleton, WI, USA) with random primers (Promega Corporation, Madison, WI, USA). Real-time PCR was performed on the CFX96 thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green I containing universal PCR master mix (A&A Biotechnology, Gdynia, Poland) and the following primers specific to mice: chemerin (5′-CTTCTCCCGTTTGGTTTGATTG, 5′-TACAGGTGGCTCTGGAGGAGTTC), SAA3 (5′-ACAGCCAAAGATGGGTCCAGTTCA, 5′-ATCGCTGATGACTTTAGCAGCCCA), cyclophilin A (5′-AGCATACAGGTCCTGGCATCTTGT, 5′-CAAAGACCACATGCTTGCCATCCA) and β-actin (5′-CCTTCTTGGGTATGGAATCCTG, 5′-TGGCATAGAGGTCTTTACGGA). The Microsoft Excel–based (Microsoft Corporation, Redmond, WA, USA) application Best-Keeper was used to analyze the expression stabilities of commonly used reference genes^{56 (link)}. Based on this analysis, murine cyclophylin A and β-actin were selected as housekeeping genes for normalizing RNA expression in RT-QPCR. Relative gene expression normalized to the geometric mean of these housekeeping genes was calculated using the 2^−ΔΔCT method^{57 (link)}.