2.5. RNA Isolation, Reverse Transcription, and RT-PCR

Total RNA was isolated from the dLN using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s directions. A QiaCube (Qiagen) automated RNA isolation machine was utilized in conjunction with the specified RNA isolation kit. The concentration and purity of the RNA was determined using a ND-1000 spectrophotometer (Thermo Scientific Nanodrop, Wilmington, DE, USA). For gene and primary miRNA expression analysis, first strand cDNA synthesis was performed using a High-Capacity cDNA Synthesis Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s recommendations. For mature miRNA, reverse transcription TaqMan MicroRNA Assays (looped-primer RT-PCR; Applied Biosystems) were utilized according to manufacturer’s recommendations (both multiplex and singleplex protocols were utilized).

For analysis of mRNA and primary miRNA expression, TaqMan Universal Fast master mix (Life Technologies, Carlsbad, CA, USA), cDNA, and mouse-specific mRNA primers (TaqMan Custom PCR Arrays, Carlsbad, CA, USA) were combined and PCR was performed according to the manufacturer’s protocol (TaqMan Gene Expression Analysis). For analysis of miRNA expression, TaqMan Universal 2× master mix, No AmpErase UNG (Life Technologies), cDNA, and mouse-specific miRNA primers (TaqMan Custom PCR Arrays) were combined and PCR was performed according to manufacturer protocol (TaqMan miRNA Assays both Single- and Multi-Plex). Primers used include: β-actin, cd25 (il2rα), ctla4, foxp3, mature miR-210, -31, -155, primary miR-210, runx3, and sno234. MicroAmp Fast Optical 96-well reaction plates were analyzed on an Applied Biosystems 7500 Fast Real Time PCR system using cycling conditions as specified by the manufacturer. β-actin (mRNA and primary miRNA) and snoRNA234 (miRNA) were used as the endogenous reference control gene as expression was determined to be stable following chemical exposure (data not shown). RT-PCR data were collected and represented as relative fold change over vehicle control, calculated by the following formula: 2^−ΔΔCt = ΔCt_Sample − ΔCt_Control. ΔCt = Ct_Target − Ct_β-ACTIN, where Ct = cycle threshold as defined by manufacturer.