Quantitect sybr green

Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s instructions. After RNA quantification (ND-1000; NanoDrop spectrometer), 1 μg of RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative RT–PCR was performed on a Corbett Rotor-Gene 6000 detection system (QIAGEN) using QuantiTect SYBR Green (QIAGEN). Relative gene expression levels were determined using the comparative quantification feature of Rotor-Gene software and normalized to GAPDH or ACTB. Primer sequences are listed in Table S2.

Table S2. Oligonucleotide and gblock sequences used for qPCR and cloning.

Culture media was removed, cells were washed with ice-cold ACSF, and RNA was isolated by phenol-chloroform extraction (TRIzol, Invitrogen). Relative transcript abundance was quantified by two-step RT-PCR (Quantitect SYBR Green, Qiagen) using the primers listed in Table 5. Primers for NFATc3 and NFATc4 (Vashishta et al., 2009 (link)), and for GAPDH (Garcia et al., 2021 (link)) were previously published and validated. Primers for Mdm2 were designed using NCBI Primer-BLAST and validated prior to use. Fold-change was calculated using the 2^−ΔΔCt method, commonly referred to as the “delta-delta Ct method”.

To assess mRNA levels of inflammatory genes in lung tissues, the total cellular RNA was extracted from the lungs using RNeasy Mini Kit, according to the manufacturer’s protocol (Qiagen Inc, Toronto, On, Canada), and reverse transcribed using SuperScript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). Real-time quantitative PCR was performed with a LightCycler System (Roche Applied Sciences, Indianapolis IN) using QuantiTect SYBR Green (Qiagen Inc, Toronto, On). Each target was quantified using four tenfold serial dilutions of standards prepared from PCR amplicons that had been gel purified and quantified using a Fluorochem 8000 imaging system and AlphaEase software (Alpha Innotech, San Jose, CA). Values were then normalized to GAPDH that had been reverse transcribed, PCR amplified, and quantified. The following mouse primers were used [19 (link)]: Muc-2, sense 5′-GCT GAC GAG TGG TTG GTG AAT G-3′ and antisense 5′-GAT GAG GTG GCA GAC AGG AGA C-3′, Muc-5ac, sense 5′-CAG CCG AGA GGA GGG TTT GAT CT-3′ and antisense 5′-AGT CTC TCT CCG CTC CTC TCA AT-3′. Eotaxin-1 (CCL11), sense 5′-GGG CAG TAA CTT CCA TCT GTC TCC-3′ and antisense 5′-CAC TTC TTC TTG GGG TCA GC-3′. GAPDH, sense 5′-GCC ATG GAC TGT GGT CAT GA-3′ and antisense 5′-TTC ACC ACC ATG GAG AAG GC-3′. IDO, sense 5′-GTA CAT CAC CAT GGC GTA TG-3′ and antisense 5′-CGA GGA AGA AGC CCT TGT C-3'.