Taqman mrna assay primers

Total RNA was collected from the cell pellet and extracted with TRI reagent (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. CCL2, CCL3, CCL4, CCL22, IL-1Rα, IL-6, IL-8, S100a9, and GAPDH levels were analyzed by qRT-PCR. mRNA qRT-PCR was performed using the TaqMan High-Capacity cDNA Reverse Transcription Kit, TaqMan Fast Advance PCR Master Mix, and TaqMan mRNA assay primers (Applied Biosystems, ?city state?). All reactions were analyzed using 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative expression of mRNA were determined by the ΔΔC_T method where the cycle threshold (C_T) values, corresponding to the PCR cycle number at which fluorescence emission reaches a threshold above baseline emission, were determined for the mRNA expression relative to untreated controls. Analyses were performed using JMP pro version 10 (SAS, Cary, NC). P-test was used to evaluate significance. P values less than 0.05 were considered significant.

Total RNA of cytokine-treated THP-1 cells or A549 cells were isolated using the mirVana Total RNA Isolation Kit (Life Technologies) following the manufacturer's protocol. RNA yield and purity were determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Wilmington, DE). Quantification of mature miRNA expression was performed using the TaqMan qRT-PCR as described [11 (link),28 (link)–30 (link)]. Equal amounts of each RNA (6.7 ng for miRNA and 33 ng for mRNA) were used for quantitative real-time RT-PCR (qRT-PCR) analysis. For mRNA analysis, a High Capacity cDNA RT Kit (Applied Biosystems) and TaqMan mRNA assay primers for mRNA expression were used. The cycle threshold (C_t) values, corresponding to the PCR cycle number at which fluorescence emission reaches a threshold above baseline emission, were determined. miRNA expression values were calculated using RNU44 as an endogenous control (Applied Biosystems) following the 2^−ΔΔCt method [31 (link)]. mRNA expression values were quantified in the same way after normalization to mammalian 18S rRNA as previously described [11 (link),12 (link),14 (link)].