Rna genelute kit

Blood samples were obtained just before experiment onset (H0), 8 hours later (H8) and at H48 (or just before death). Blood, lung and spleen concentrations of IL-8, IL-1ß, IL-10 and TNF-α were assessed by enzyme-linked immunosorbent assay (ELISA) (Euromedex). Lung and spleen pieces were taken and RNA was extracted using the RNA GenElute kit (Sigma). Complementary DNA (cDNA) was obtained by reverse transcription using random primers, RNAsin treatment, and ImProm II reverse transcriptase (Promega, Madison, WI). Quantitative PCR was then performed using the IQ5 thermocycler (Biorad, Hercules, CA) and the IQ^TM Syber Green Supermix (Biorad) and rabbit-specific primers, designed using Primer3 software (version 0.4.0), and the rabbit (Oryctolagus cuniculus) sequence database. Melting curves were performed to ensure the presence of a single amplicon. The primers sequences for Gapdh, Il-8, Il-1β, were reported in Table S1. The results are expressed as the fold induction using the ΔCt method since the spontaneously breathing (SB) animals were always considered the baseline condition.

RNA was extracted from lung samples using the RNA GenElute kit (Sigma-Aldrich, St Louis, MO, USA). Quantitative PCR was performed using IQ Sybergreen Supermix (Bio-Rad, Hercules, CA, USA). Melting curves were performed to ensure the presence of a single amplicon. The following primers were used: rTlr2, forward 5’-TGT CTG TCA CCG AAC CGA ATC CAC-3’ and reverse 5’-TCA GGT TTT TCA GCG TCA GCA GGG-3’ [20 (link)]; and rGapdh, forward 5’-ATG TTT GTG ATG GGC GTG AAC C-3’ and reverse 5’-CCC AGC ATC GAA GGT AGA GGA-3’.
The results are expressed as the fold induction using the ΔCt method since the SB animals were considered as the baseline condition.

All gene expression analyses were done 8 hours after lung exposure to either saline, Pam3CysSerLys4 (Pam₃CSK₄) or S. aureus, since peak values were achieved within this timeframe in previous studies [19 (link)]. RNA was extracted from lung samples using the RNA GenElute kit (Sigma-Aldrich, St Louis, MO, USA). Quantitative PCR was performed using IQ Sybergreen Supermix (Bio-Rad, Hercules, CA, USA). Melting curves were performed to ensure the presence of a single amplicon. The following primers were used: rTlr2, forward 5’-TGT CTG TCA CCG AAC CGA ATC CAC-3’ and reverse 5’-TCA GGT TTT TCA GCG TCA GCA GGG-3’ [23 (link)]; rIl-8, forward 5’-AAC CTT CCT GCT GCT TCT GA-3’ and reverse 5’-TCT GCA CCC ACT TTT TCC TTG-3’; TNFα, forward 5’-CAA GCC TCT AGC CCA CGT A-3’ and reverse 5’- GGC AAT GAT CCC AAA GTA G-3’; and rGapdh, forward 5’-ATG TTT GTG ATG GGC GTG AAC C-3’ and reverse 5’-CCC AGC ATC GAA GGT AGA GGA-3’.
The results are expressed as the fold induction using the ΔCt method [24 (link)] since the SB animals were always considered as the baseline condition.