Total RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA extraction and qRT-PCR were performed as described before⁵². Briefly, total RNA was extracted using the NZYol (NZYTECH, Lisbon, Portugal) and quantified in a NanoDrop ND-1000 spectrophotometer at 260 nm. RNA purity was assessed by analysis of 260/230 and 260/280 absorption ratios. The cDNA was reverse-transcribed using NZY First Strand cDNA Synthesis Kit (NZYTECH), beginning with 2 µg of total RNA. qRT-PCR was performed, in duplicate for each sample, using NZYSpeedy qPCR Green Master Mix (2×) (NZYTECH) on CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).

The efficiency of the amplification reaction for each gene was calculated using a standard curve of a series of diluted cDNA samples and the specificity of the amplification products was assessed by analysing the melting curve generated in the process.

Gene expression changes were analysed using the built-in CFX Manager software which enables the analysis of the results by the Pfaffl method, a variation of the ΔΔCT method corrected for gene-specific efficiencies⁵³. The results were normalized using Hprt1 as housekeeping gene. This gene was experimentally determined with Genex software using NormFinder and geNorm algorithms (MultiD Analyses AB, Göteberg, Sweden) as the most stable for the treatment conditions used. Specific sets of primers for Nos2, Il1b and Hprt1 (Table 4) were designed using Beacon Designer software version 8 (Premier Biosoft International, Palo Alto, CA, USA).

Oligonucleotide Primer Pairs Used for qRT-PCR.