Lightcycler 480 sybr green 1 master

RNA extraction was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA samples were extracted from 5 birds tissues and used as a template for reverse transcription. cDNA was obtained by reverse transcriptase SuperScript® III First-Strand Synthesis System using 2 μg total RNA. The relative abundance of mRNA from genes was assessed by real time reverse-transcription (RT)-PCR using a Lightcycler 480 (Bio-Rad) and a Lightcycler 480 SYBR Green I master (Bio-Rad). Primer pairs specific for the amplification of AvBD, cathelicidin and NK-lysin genes are shown in Additional file 1: Table S1. PCR products were subjected to melt curve analysis and sequenced to confirm amplification of the correct gene. Data were analyzed by ddCt method. The mean threshold cycle value (Ct) of each sample was normalized to the internal control, GAPDH, and the expression profiles were obtained by comparing normalized Ct value with the calibrator sample, in which the gene exhibited the lowest expression level. Each analysis was performed in triplicate. Quantification of each sample was calculated with the cycle threshold values and standard curve information using the Lightcycler 480 version 1.5.0 software.

mRNA expression levels were evaluated via real-time quantitative PCR (qPCR) using the LightCycler 480 SYBR Green I Master and the SYBR Green I (483–533 nm) detection format on a CFX96 (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer’s instructions. The primer pairs used to perform qPCR were as follows: TERT - forward primer (P570) located in exon 6 and reverse primer (P571) located in exon 7; and EZH2 - forward primer (P563) located in exon 2 and reverse primer (P564) located in exon 3. The expression level of H6PD, determined via the primer pair, (P114) and (P115), was used as an internal reference for normalization. PCR conditions were as follows; 95 °C for 5 min, 45 cycles of 10 s at 95 °C each, 55 °C for 10 s and 72 °C for 10 s. A standard curve was generated using serially diluted cloned PCR products of both the internal reference and target genes. Expression was measured relative to the human total brain RNA (Clontech Laboratories, Mountain View, CA, USA). Primer sequences are described (Additional file 1 Table S1).

Cells (NDF, HaCaT, and HUVEC) were seeded on the 6‐well plate without any stimulation at a density of 2 × 10⁴ and cultured to reach the confluency of 80%. Cells were then harvested for the extraction of total RNA using PureLink RNA Mini Kit (Thermo Fisher Scientific). 1 µg RNA was reverse transcribed using qScript cDNA SuperMix (Quanta BioSciences). Real‐time qPCR was performed on the cDNA with Light‐Cycler480 SYBR Green I Master on a CFX96 Touch System (Bio‐Rad). Primers were purchased from Integrated DNA Technologies (Table S3, Supporting Information). mRNA expression was normalized to the GAPDH expression using the 2^−∆∆Ct method. Each experiment was performed three times in duplicate. For the growth factor stimulation study, cells were first starved for 24 h in DMEM with 1% FBS before the addition of growth factors for 2 days.