Quantstudio 6 flex software

HMEECs were seeded in 96-well plates at 1.7 × 10⁴ cells/well. After 24 h, they were incubated with 0, 1.5, and 2 mM FA for 4 h and 0, 0.1, and 0.2 mM FA for 24 h. Total RNA was extracted from the HMEECs using an RNeasy® mini kit (Qiagen GmbH, Hilden, Germany). RNA (3 μg) was reverse-transcribed at 55°C for 30 min and the resulting cDNAs were then amplified using a PCR kit (Qiagen, Valencia, CA, USA). The sequences of the oligonucleotide primers used in the PCRs were as follows: TNF, 5′-GGAGAAGGGTGACCGACTCA-3′; MUC5AC, 5′-CAGCACAACCCCTGTTTCAAA-3′; and GAPDH, 5′-ATGGCACCGTCAAGGCTGAG-3′. PCR amplification was performed under the following conditions: a holding stage at 95°C for 20 s followed by 45 cycles of 95°C for 1 s and 60°C for 20 s and then a melting stage at 95°C for 15 s and 60°C for 60 s. The results were obtained from three repeated experiments using triplicate samples. The data were analyzed using QuantStudio 6 Flex software (Applied Biosystems, Foster City, CA, USA).

One microliter of cDNA was used as a template for real-time PCR with primers (Table 1). PCR was carried out with 10-μl reactions, prepared as follows: 5 μl of 2 × SYBR premix Ex SYBR, 0.2 μl of each primer (10 mM), 0.2 μl of ROX reference dye II, 1.0 μl of cDNA template, and 3.4 μl of RNase-free water. A QuantStudio 6 Flex system (Applied Biosystems, Foster City, CA, United States) was applied with the following protocol: initial denaturation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 30 s and annealing at 60°C for 30 s; and a final melting curve program of 15 s at 95°C, 1 min at 60°C, and 15 s at 95°C. The relative expression of the tested sRNAs (including CyaR, MicC, InvR, RybB, MicA, and DsrA) in comparison with 16S DNA was analyzed using QuantStudio 6 Flex software from Applied Biosystems. The 2^–ΔΔCt method was used to determine the relative expression of each sRNA in comparison with the 16S rRNA gene as an internal control, where ΔΔCt = (C_t, _sRNA − C_t, _16SrRNA)_stress − (C_t, _sRNA − C_t, _16SrRNA)_control. The relative fold change in the expression of genes was determined using five replicates. The relative levels of each sRNA were determined.