Bryt green

Thymic tissue was lysed for RNA extraction by the RNeasy Plus Mini Kit (Qiagen, Valencia, California) in accordance with manufacturer instructions. The total RNA concentration was measured by spectrophotometry in a Nanodrop ND1000 system, and the first‐strand cDNA was synthesized from total RNA using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, California). Relative mRNA levels were measured with Bryt Green (Promega, Fitchburg, Wisconsin) using a Mastercycler ep realplex PCR system (Eppendorf, Hamburg, Germany). All experiments were performed in triplicate. The relative level of each gene was normalized to the housekeeping gene acidic ribosomal phosphoprotein P0 (36B4) and expressed as the fold change relative to the CTRL group using the 2^−ΔΔCt method, where ΔCt = Ct_{(target gene)} − Ct_{(housekeeping gene).}16 The mRNA expression of the following genes was analyzed: indoleamine 2,3‐dioxygenase (IDO)‐2, CD39, galectin, cytotoxic T‐lymphocyte‐associated antigen (CTLA)‐4, PD‐1, and IL‐10. The sequences of PCR primers can be found in Supporting Information Table S1.

To isolate miRNA-containing RNAs, cells were lysed and homogenized with Acid Phenol (Ambion®, Life Technologies, Darmstadt, Germany) and RNA was isolated using the miRVana RNA isolation kit (Ambion®, Life Technologies, Darmstadt, Germany) according to the instructions provided by the manufacturer. RNA concentration was measured using the Nanodrop 1000 (Thermo Scientific, Waltham, USA) and RNA integrity was assessed using the Bioanalyzer (Agilent, Santa Clara, USA).
cDNA synthesis and primer design (Tab. 2) for the analysis of miRNA expression, profiling by miQPCR was carried out as previously described31 (link). In brief, 10 ng of total RNA was used for cDNA synthesis and a fraction of the synthesized cDNA (equivalent to 1% of final cDNA volume) was used for individual qPCR assays. Primer design was performed as described in31 (link), miRNA specific primers used in this study are listed in Table 2. qPCR assays were performed in a 96 well format and run on a ViiA7 thermocycler (Applied Biosystems®, Life Technologies, Darmstadt, Germany) using Bryt^® Green (Promega, Mannheim, Germany) miRNA amplification parameters were as follows: 95 °C for 10 minutes (1 cycle), 95 °C for 15 seconds and 60 °C for 35 seconds (50 cycles), followed by melting curve analysis. qPCR data were analyzed by qBase software v.1.3.546 (link).

Total RNA was extracted from homogenized lung cells with Tri-reagent (Sigma-Aldrich), according to the manufacturer’s instructions 24 h after the last instillation. cDNA was synthesized using the Promega GoScript Reverse Transcription System. RT-qPCR was performed on a Stratagene Mx 3000P using a qPCR Go Taq master mix with Bryt Green (Promega). Each RT-qPCR amplification was performed in duplicate under the following conditions: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The forward and reverse primers used for IL-4 were 5-GTGCAGCTTATCGATGAATCC-3′ and 5-AGCCATATCCACGGATGCGAC-3, Hydroxymethylbilane synthase (HmBS, forward 5′-GAAACTCTGCTTCGCTGCATT-3′, reverse 5′-TGCCCATCTTTCATCACTGTATG-3′) mRNA was used as the reference housekeeping gene for normalization. For each sample (x), the normalization factor was calculated using the formula, mean of Ct of ref(i) − Ct of ref(x), where i represents all samples (Ct is on the threshold cycle-value shown as the mean of three different RT-qPCR reactions), as described in Ref. (34 (link)). The level of target mRNA, relative to the mean of the reference housekeeping gene, was calculated by raising 2 to the power of {40 − [Ct of target + mean of (Ct of norm. HmBS)]}.