Colour reverse transcription kit

Total RNA was extracted from HRECs and retinas using a universal RNA Purification Kit (B004, EZ Bioscience, USA) following the manufacturer’s protocol. The concentration and quality of RNA were examined using Nanodrop 2000 (Thermo Fisher Scientific). RNA samples were reverse-transcribed to complementary DNA (cDNA) using a Colour Reverse Transcription Kit (A0010CGQ, EZBioscience). Quantitative polymerase chain reaction (qPCR) was performed using SYBR Green Master Mix (A0012, EZBioscience). GAPDH was used as an internal reference for each reaction. The relative expression was calculated using the following equation: Relative gene expression = 2^{[△Ct(control)–△Ct(target)]}. The sequences of the primers are listed in Additional file 1: Table S1.

Cell culture of RAW264.7 and HaCaTs were as aforementioned for RNA extraction. Total RNA was extracted from the samples using the EZ-press RNA Purification Kit (EZBioscience, Roseville, MN, USA). Following extraction, the RNA was reverse transcribed into complementary DNA (cDNA) utilizing the Colour Reverse Transcription Kit (EZBioscience). Quantitative RT-PCR was then performed using the 2 × Color SYBR Green qPCR Master Mix (EZBioscience). The relative expression levels of the target genes were calculated employing the 2^−ΔΔCt method (n = 3). Primer sequences are shown in Table S1 (Additional file), and GAPDH was used as internal reference gene.

RAW264.7 cells treated with different CM for 24 h were evaluated for iNOS and Arg-1 expression by RT-PCR. Total RNA was extracted and purified using the EZ-press RNA purification kit (EZBioscience, Roseville, MN, USA) and then reverse transcribed into cDNA using a Colour reverse transcription kit (EZBioscience). Quantitative RT-PCR was performed using 2 × Colour SYBR Green qPCR master mix (EZBioscience) and relative expression was calculated using the 2^−ΔΔCt method. Primer sequences for glyceraldehyde 3-phiosphate
dehydrogenase (Gapdh), iNOS, and Arg-1 are shown in Additional file 1: Table S1.