Taqman gene expression assays primers probe sets

Twenty-four hours post-exposure, we collected the BEAS-2B cells, which were pooled from the three technical replicates, for subsequent RNA extraction. As previously described [20 (link)], we conducted RNA isolation using the Qiagen RNeasy Micro Kit (catalog number 74004) with Trizol/chloroform extraction. We used a NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA) to determine the RNA concentration and purity. cDNA was prepared using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) and amplified with a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). We used either designed primers, as listed in Table 1 or inventoried TaqMan gene expression assays primers-probe sets (Applied Biosystems, Waltham, MA, USA) to conduct quantitative real-time PCR (Applied Biosystems 7300 Real Time PCR System) on cDNA samples. We used the 2−ΔΔCt method to calculate fold-changes of exposed groups compared to air controls. For normalization, we used β-ACTIN as the housekeeping gene. We expressed the results as fold-change over air controls.

Isolation and processing of RNA from pooled H292 cells were performed according to procedures described previously [37 (link)]. Briefly, total RNA was isolated by a TRIzol/chloroform extraction, followed by column purification with the Qiagen RNeasy Mini Kit. RNA concentrations were measured with a ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE). Isolated pure RNA was reverse transcribed using iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA). The resulting cDNA was amplified in a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). Quantitative real-time PCR was performed on cDNA samples from H292 cells with either inventoried TaqMan gene expression assays primers-probe sets (Applied Biosystems) or designed primers listed and described in Additional file 1: Table S1. Reaction volumes were 25 µL and reaction cycles were performed for each gene in an Applied Biosystems 7300 Real Time PCR System. Fold changes were calculated using the 2−ΔΔCt method. β-ACTIN was the housekeeping gene used for normalization. Results are reported as fold-change over air-control group. A fold-change > ± 1.5 was considered significant.