Taqman gene expression assay reagent

The reverse transcription reaction was performed using an AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA). Briefly, total RNA (3 μg) was reverse transcribed using random primers according to the manufacturer's instructions. cDNA aliquots were used for quantitative PCR analysis. Real-time PCR was performed using TaqMan probes with Brilliant II QPCR Master Mix (Agilent) according to the manufacturer's instructions. TaqMan Gene Expression Assay reagents (Thermo Fisher Scientific, Waltham, MA, USA) for aromatase (Assay ID: Mm00484049_m1) and β-actin (Assay ID: Mm01205647_g1) were used as TaqMan probes. Real-time PCR was performed using a two-step cycling protocol consisting of 45 cycles of 20 s at 95°C and 60 s at 60°C on an Mx3000P QPCR System (Agilent). All reactions included controls lacking the template. After the reactions, the Ct values were determined using fixed-threshold settings. The ΔΔCT method was used to determine the mRNA fold change, which was normalized to β-actin mRNA level. Each experiment was performed in duplicate and repeated at least three times.

Gene expression was measured using quantitative real-time PCR real-time fluorescence detection. TaqMan Gene Expression Assay reagents and TaqMan FAM dye-labeled probes (Thermo Fisher Scientific) were used to set up appropriate reactions according to the manufacturer’s protocol, using PCR Master Mix (2×) (Thermo Fisher Scientific K0171) and the ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems). To determine the most suitable housekeeping gene to use, we performed GeNORM analysis (kit from PrimerDesign) of 12 housekeeping genes in advance. Canx was determined as the most stable gene between samples and conditions and used as a housekeeping gene. The following TaqMan probes were used (gene name, TaqMan assay, and TaqMan assay ID): Canx: Mm00500330_m1, Rpgrip1l: Mm00452421_m1, Fto: Mm00488755_m1, Irx3: Mm00500463_m1, Irx5: Mm00502107_m1, Irx6: Mm01253620_m1, Ppargc1a: Mm01208835_m1, Ucp1: Mm01244861_m1, Cox8b: Mm00432648_m1, Prdm16: Mm00712556_m1, Dio2: Mm00515664_m1, Elovl3: Mm00468164_m1, Pparg: Mm00440940_m1, Cebpa: Mm00514283_s1, Fabp4: Mm00445878_m1, Plin1: Mm00558672_m1, Fasn: Mm00662319_m1, Adrb3: Mm02601819_g1, and Cox7a1: Mm00438297_g1.

Total mRNA was extracted from the spleen and thymus. The isolated total mRNA was suspended in DEPC water for storage at −70°C and cDNA synthesis was performed with First Strand cDNA synthesis kit purchased from Pharmacia Biotech. The amplification of Ifng, Nos2, Il4, Arg1, Foxp3, Il10, Tgfb, Ebi3, Il12a, Il12b, Il17 and 18S cDNAs was performed in 384-well MicroAmp optical reaction plates using commercially available primer and probe sequences (TaqMan Gene Expression Assay reagents) and Universal PCR Master Mix (all from Applied Biosystems, Forter City, CA, USA). The total reaction volume in each well was 10 μl containing 5 ng of cDNA. The reactions were carried out in AB7900 HT Sequence Detection System (Applied Biosystems) using the standard three-step run protocol (Step 1: 2 min at 50°C, Step 2: 10 min at 95°C, Step 2: 40 cycles of 15 s at 95°C plus 1 min at 60°C). Quantification was calculated by the comparative threshold cycle (C_T) method following the manufacturer’s instructions. All quantifications were normalized to the 18S gene to account for the variability in the initial mRNA concentration the conversion efficiency of the reverse transcription reaction (ΔC_T) as well as values from control samples from non-infected rats (ΔΔC_T). The relative quantity (RQ) was calculated as: RQ = 2^-ΔΔCT. SDS 2.2.2 software was used for analysis.

Total RNA was isolated from individual samples by using RNeasy Plus Mini Kit (Qiagen, Hilden Germany). Reverse transcription was performed by using the High-Capacity cDNA Reverse Transcription Kit and quantitative real-time PCR was done on a 7500 Fast Real-Time PCR System instrument using Taqman Fast Universal PCR Master Mix (2x) and No AmpEraseUNG (all from Applied Biosystems, Foster City, CA). Probes and primers were from Taqman gene expression assay reagents (Applied Biosystems). Data were normalized to Mrpl 32 (Mm00777741-sH) RNA amplification and calculated relative to the expression of the target gene in freshly isolated T cells.