Bioline tetro cdna synthesis kit

Bone marrow was harvested from the tibia and femur, using Iscove's Modified Dulbecco's Medium (IMDM). BM was incubated with 10% foetal calf serum (FCS) and stem cell factor (SCF, 100 ng mL⁻¹) in IMDM for 2 h to enrich for HSPC populations. The supernatant was acquired, washed with PBS and processed for RNA extraction using TRIzol, and cDNA synthesised using Bioline Tetro cDNA synthesis Kit (catalogue number BIO‐65043), both as per manufacturer's instructions.

The RNA samples were isolated by applying trypsin (Sigma-Aldrich) to the AOH/AME/LPS-treated BEAS-2B or RAW264.7 cells and RNA was extracted using mammalian RNA extraction kits and manufacturer protocols (Qiagen, Valencia, CA, USA). The samples were processed into cDNA following the manufacturer’s instructions from the Bioline Tetro cDNA synthesis kit, and then stored at −20 °C (Bioline, London, UK). All the qRT-PCR reactions for the biological triplicates were performed as technical duplicates using the cDNA as a template. GAPDH was used as a control housekeeping gene for all experiments, as it has a continuous expression in mammalian cell lines. A BIO-RAD Iq5 Multicolor Real-Time PCR Detection System machine was used to conduct the qRT-PCR reaction (Bio-Rad, Hercules, CA, USA). All reactions were carried out at 20 μL volume with SYBR Green (Bioline) as the fluorescent reporter molecule. Relative fold change in gene expression was calculated using the 2(-Delta Delta C(T)) method and Pfaffl equation by normalization to GAPDH, as suggested by Bio-Rad protocols.

RNA was extracted using Qiagen RNEasy kits (Qiagen, Machester, UK) and assessed for purity using a NanoDrop ND‐1000 (Thermo Scientific, Wilmington, DE). cDNA was synthesized using the Bioline Tetro cDNA synthesis kit (Bioline, London, UK). All TaqMan primer/probes were obtained from Thermo Fisher Scientific. The following primers were used in this study: CCL20 (Hs00355476_m1), FOXP3 (Hs01085834_m1), GAPDH (Hs02758991_g1), IL‐1β (Hs00174097_m1), IL‐6 (Hs00985639_m1), SHP (Hs00222677_m1). Primer information is detailed further in the supplementary material. Each reaction was run for 40 cycles on a StepOnePlus real‐time PCR machine (Thermo Fisher Scientific) using SensiFAST probe Hi ROX kit (Bioline).