Taqman chemistry

Quantitive gene expression analysis was performed by means of quantitative Polymerase Chain Reaction (qPCR) with TaqMan chemistry (Life Technologies, CA, USA). A total of 1 ng/reaction cDNA together with Master Mix was added to all genes simultaneously using a Nanodrop II dispenser (GC biotech, Netherlands), for a final reaction volume of 2 μl per gene and sample. QPCR reaction was run on the real-time PCR, ABI PRISM 7900HT Sequence Detection System (Life Technologies, CA, USA). Raw data was analyzed with the SDS 2.4 and RQ manager 1.2.1 software provided by the instrument.

As above, brains from each group were collected 10 min after the end of a 10-min FST (N = 4–5/group/treatment), and frozen immediately on dry ice. Brain regions of interest were punched using 0.5 mm ID biopsy corer (Fine Science Tools, Foster City, CA, USA) and kept at −80°C until extraction. mRNA isolation was performed using Qiazol (Trizol-chloroform) extraction with RNeasy column clean-up (Qiagen, Valencia, CA, USA). Samples were stored at −80°C in 1 mM sodium citrate, pH 6.4 (Life Technologies, Grand Island, NY, USA) prior to cDNA synthesis. mRNA concentrations were measured using spectrophotometry and diluted to the same concentration for all samples. cDNA synthesis was preformed with MultiScribe™ MuLV reverse-transcriptase following the protocol for the high capacity cDNA synthesis kit (Life Technologies). cDNA was stored at −20°C. cDNA synthesis reaction was preformed in triplicate for vHC samples. Assays were performed using the TaqMan chemistry and off the shelf assays from Life Technologies. Assay IDs were: Gapdh-Mm99999915_g1 and cFos-Mm00487425_m1. Samples were run in triplicate on a Life Technologies 7900HT real-time PCR machine with a 20 μl reaction volume. Samples were compared using the ΔΔC_T method of relative quantification. GAPDH was used to normalize between biological replicates.

Total RNA was isolated using RiboPure Kit or Trizol reagent (both from Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The purity and concentration of RNA was measured with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). Five-hundred nanograms of total RNA were used for cDNA synthesis using random hexamer primers (Promega, Madison, WI, USA) and Maxima reverse transcriptase (Fermentas, Thermo Fisher Scientific). The relative expression levels of mRNA were measured according to the manufacturer's protocol by qRT-PCR (StepOnePlus; Life Technologies Carlsbad, CA, USA) using TaqMan chemistry and specific assays-on-demand target mixes (Life Technologies, Carlsbad, CA, USA). The expression levels were obtained by normalizing the target gene to ribosomal RNA or to GAPDH, and presented as fold change in the expression or in arbitrary units using the 2^-ΔΔCt method; Ct is the threshold-cycle value.

Wing punch biopsy samples were collected directly into tissue lysis buffer with proteinase K (DNeasy Blood & Tissue Kit, Qiagen, Halden, Germany) and isolated within 10 h of sampling according to the manufacturer’s protocol. Fungal load in the samples was estimated using a qPCR method based on TaqMan chemistry (Life Technologies, Foster City, CA, USA) and using dual probes for P. destructans and Pseudogymnoascus sp. detection50 .

Viral RNA (vRNA) was extracted from all samples using MagNA PURE 96 (Roche, Mannheim, Germany), following the manufacturer’s instructions. All samples were then tested for the presence of influenza viruses (types A and B) and respiratory syncytial virus (RSV), in a single real-time reverse transcriptase polymerase chain reaction (RT-PCR) performed using Ambion Ag-Path master mix (Thermo Fisher Scientific, Waltham, MA, USA) and TaqMan Chemistry (qRT-PCR), on an ABI 7500 platform, as described previously [19 (link)].