Superarray pcr master mix

PMs were infected with N. caninum at a multiplicity of infection (MOI) of 3:1 (parasite:cell) for 12 h and 24 h and harvested with 1 ml of TRIzol reagent (Life Technologies, Carlsbad, USA) for total RNA extraction according to the manufacturer’s instructions. After quantification using a Nanodrop ND-2000 apparatus (Thermo Scientific, Wilmington, USA), 2 μg of total RNA was used for cDNA synthesis using the RT² First Strand Kit (SA Biosciences/Qiagen, Shanghai, China).
The 96-well Mouse Inflammasome RT Profiler PCR Arrays (SA Biosciences/Qiagen, Shanghai, China), containing primer pairs for 84 key genes involved in the NLR inflammasome pathway, were probed with cDNA template in the presence of SuperArray PCR master mix (Qiagen, Shanghai, China), and the PCR analysis was performed on a Bio-Rad iCycler (USA). The arrays were probed with cDNA from 3 treatments (12 h, 24 h and the control group) in duplicate, and the results were analyzed by Excel-based PCR Array Data Analysis software (SA Biosciences, Shanghai, China) for threshold cycle (Ct) value determination. The Ct of the genes was normalized to that of housekeeping genes (the average Ct of Actb, B2m, Gapdh and Hsp90ab1). The relative expression level of each target gene in infected cells was calculated as 2^-ΔΔCt (fold change), where ΔΔCt represents the Ct (sample) - Ct (control).

The Human Autophagy RT2 Profiler PCR Array (SABiosciences, PAHS-084Z) was used to study autophagy-specific gene expression profiles in accordance with the manufacturer's recommendations. Briefly, total RNA was isolated from different experimental groups using Trizol (Invitrogen, 15596-018). Potential genomic DNA contamination was removed from samples by treatment with RNase-free DNase (Invitrogen, 79254) for 15 min at 37°C. The RNA concentration and purity were determined using a NanoDrop ND-1000 (Thermo Scientific). First-strand cDNA was synthesized from 1–2 μg total RNA using SuperScript III Reverse Transcriptase (Invitrogen, 18080044). After cDNA synthesis, real-time PCR was performed using SuperArray PCR master mix (Qiagen, 330503) and a Roche LightCycler® 480 (96-well Block) according to the manufacturer's instructions. Amplification data (fold-changes in Ct values of all genes) were analyzed using the ΔΔCt method.