Oligo dt and random primers

Macrophages were incubated with H. polygyrus antigens at a concentration of 10 µg/mL for 24 h in standard conditions (37 °C, 5% CO₂). After incubation, the culture medium was removed, and the cells were lysed using Fenozol Plus solution (A&A Biotechnology, Gdansk, Poland) using the RNA Total RNA Mini Plus isolation kit. The cell suspensions were then transferred to tubes and stored at −21 °C until RNA isolation. The experiment was performed three times.
Total RNA was isolated from macrophages using the RNA Total RNA Mini Plus kit (A&A Biotechnology) according to the manufacturer’s protocol. cDNA was then synthesized using the FIREScript RT cDNA synthesis mix with oligo (dT) and random primers (Solis BioDyne, Tartu, Estonia). The qPCR was performed using the SYBR Select Master Mix kit (Thermo Fisher Scientific) and 96-well plates from Roche (Basel, Switzerland) on a LightCycler 96 thermal cycler.
The primers were purchased from Oligo (Warsaw, Poland) (sequences can be found in the Supplementary Materials, Table S1). In this study, primers for genes coding for the following molecules were used: Arg1 (Arginase), iNOS (Nitric Oxide Synthase), YM1, IL-4, IL-10, IL-6, TNF-α, CD206, and CCL2 (chemokine (C-C motif) ligand 2). PPIA (Peptidylprolyl isomerase A) was used as the reference gene.

Total RNA was isolated from adipocytes with NucleoSpin RNA (MACHEREY-NAGEL, Dueren, Germany) according to the manufacturer’s protocol. The amount and quality of isolated RNA was measured on spectrophotometer NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA). Total RNA (1 µg) was used for reverse transcription with FIREScript RT cDNA Synthesis MIX with Oligo (dT) and random primers (Solis BioDyne). A negative control (RT-) lacking reverse transcriptase was also prepared. Resulting cDNA was used for quantitative RT-PCR (qPCR) using 5x HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (Solis BioDyne) and specific primers for leptin, insulin receptor and glucose transporter, interleukin 6 and TNFα [18 (link)]. PCR was run on Quant Studio 12K Flex system (Thermo Fisher Scientific, USA). The expression was analyzed using the comparative ∆∆Ct method. Melting curve analysis was done to verify the specificity of PCR product. For each sample, the expression levels of the target genes were normalized to the reference gene (Gapdh). Negative and positive control of reverse transcription and qPCR reactions were also performed.