Exicycler 96 real time pcr system

Real-time PCR assay was assessed to examine the expression of neurogenesis-related genes. The specimen for each groups were placed into a 24-well plate and then Schwann cells with a density of 2 × 10⁵ cells per well were seeded on the surface of various disks under a humidified atmosphere of 5% CO₂ at 37 °C. After culturing for 1, 4 and 7 days, total RNA was extracted from treated cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using a Revert Aid kit (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. PCR reactions were setup using SYBR Premix Ex Taq (Takara Bio) and performed with an Exicycler 96 real-time PCR system (Bioneer, Daejeon, Korea) [33 (link), 34 (link)]. The mRNA level of cells cultured on pure titanium foils was set as the baseline. Fold changes in mRNA levels were calculated using the 2^−ΔΔCt method. Table 1 shows the primer sequences.Table 1

Primer sequences used in the qRT-PCR Experiments

Gene	Primer sequences
GDNF	5′-CAGAGGGAAAGGTCGCAGAG-3′
	3′-ATCAGTTCCTCCTTGGTTTCGTAG-5′
NGF	5′-TCAACAGGACTCACAGGAGCA-3′
	3′-GGTCTTATCTCCAACCCACACAC-5′
GAPDH	5′-GGCACAGTCAAGGCTGAGAATG-3′
	3′-ATGGTGGTGAAGACGCCAGTA-5′

Using the Total RNA Isolation Kit (Tiangen Biotech Co. Ltd., Beijing, China) and BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China), total RNA was isolated from the lungs and reverse transcribed into the first cDNA. A quantitative PCR assay was carried out by Exicycler™ 96 Real-time PCR System (Bioneer Corporation, Daejeon, Korea) using SYBR Green (Solarbio, Beijing, China). The level of mRNA was normalized to GAPDH. The primers were synthesized by Genscript Biotechnology Co., Ltd. (Nanjing, China), and the sequences were shown as follows: human PTPRO forward 5′-CTGACCTGCCAGAAACAA-3′, reverse 5′-AGGACCCAAAGGATAGAG-3′; rat PTPRO forward 5′-TGCTCGGGCTCTTTGTGC-3′, reverse 5′-ATCGGGATGGTTTGGTGA-3′; TNF-α forward 5′-CGGAAAGCATGATCCGAGAT-3′, reverse 5′-AGACAGAAGAGCGTGGTGGC-3′; IL-6 forward 5′-AACTCCATCTGCCCTTCA-3′, reverse 5′-CTGTTGTGGGTGGTATCCTC-3′; KC forward 5′-ACCCAAACCGAAGTCATAGC-3′, reverse 5’-GGGACACCCTTTAGCATCTT-3′; MCP-1 forward 5′-CTGTCACGCTTCTGGG-3′, reverse 5′-GCCGACTCATTGGGAT-3′; MIP-2 forward 5′-ACTGGTCCTGCTCCTCCT-3′, reverse 5′-TTAGCCTTGCCTTTGTTC-3′; OCC forward 5′-CAGAGCCTATGGAACGG-3′, reverse 5′-CAAGGAAGCGATGAAGC-3′; ZO-1 forward 5′-ATCTCCAGTCCCTTACCTTTC-3′, reverse 5′-TGGTGCTCCTAAACAATCAG-3′.

A549 cells at 2 × 10⁶ cells/well in 6-well plates (Nunc™) were pre-treated with various concentrations of the Pug-4 peptide (25–100 µM) for 1 h at 37 °C in 5% CO₂ and infected with NTHi at a MOI of 10. Following a 9-h incubation, the total RNA was isolated from A549 cells using TRIzol reagent (Invitrogen, Waltham, MA, USA) and the total RNA quantity was evaluated using a Nanodrop spectrophotometer (NanoDrop Technologies, USA). Thereafter, 2 µg of extracted RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific) as recommended by the manufacturer. The qPCR was performed using the SYBR GreenStar qPCR Master Mix (Bioneer, Daejeon, South Korea) and specific oligonucleotide primers for the genes IL-1β, TNF-α, iNOS and COX-2 (Table 1). All primers were synthesized by the Integrated DNA Technologies (IDT), Canada. Reactions were amplified and quantified in an Exicycler™ 96 Real-Time PCR System (Bioneer, Korea). Thermal cycles were completed at 95 °C for 2.5 min followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s (for IL-1β, TNF-α and iNOS) and 40 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s (for COX-2 and gapdh). The comparative threshold cycle (Ct) method was used to obtain relative quantities of mRNAs which were then normalized against gapdh as an endogenous control.