Rt2 sybr green rox fast mastermix

qRT-PCR was used to confirm the expression of mRNAs and lncRNAs in microglia. First-strand cDNA was synthesized from total RNA (1 μg) using the SuperScript III First Strand Synthesis Super System (Invitrogen). qRT-PCR was performed on an ABI QuantStudio3 system (Applied Biosystems, Foster City, CA, USA) using the RT² SYBR Green ROX FAST Mastermix (Qiagen). The amplification conditions were 95 °C for 10 min, then 40 cycles at 95 °C for 15 s and 60 °C for 1 min. All of the primers used are listed in Additional file 2. Gene expression was normalized to GAPDH mRNA. Expression was calculated using the 2^−∆∆CT method.

The expression of sXBP1, RFX1, TAP1, IL18, and BCAP31 was monitored by qPCR using RT² SYBR Green ROX FAST Mastermix (Qiagen) with primers listed in Table 1, in a RotorGene 6000 instrument (Corbett life science, Sydney, Australia). The amount corresponding to 2 ng of total RNA used for cDNA synthesis was loaded into each PCR tube. For all targets, the amplification conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 50 s. A non-template control was included in duplicate for each primer pair reaction as negative control. To exclude the presence of non-specific products or primer dimers, a melting curve analysis from 65 to 95°C was performed at the end of each run. B2M (beta-2-microglobulin) and/or GUSB (Beta-D-Glucuronidase) were used as reference genes. In order to evaluate the expression of three microRNAs (miR-126, miR-146a, and miR-346), the qPCR analyses were performed on a 7500 Real Time PCR System (Applied Biosystems) using specific Taqman small RNA assays (Applied Biosystems). The reactions were performed in a final volume of 20 μl with the following thermal protocol: 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 1 min. The data were normalized against small nucleolar RNAs (RNU44 and/or RNU48). The relative expression levels were calculated using the 2^-ΔΔC_t method (Pfaffl, 2001 (link)).

Total RNA was isolated using Trizol (Thermo Fisher) according to the manufacturer’s protocol. RNA concentration was determined using a NanoDrop (Thermo Fisher) and equal amounts of RNA were added to cDNA synthesis reactions. cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Thermo Fisher) according to the manufacturer’s protocol. Real-time PCR was performed on an ABI Prism 7900HT sequence detection system using RT² SYBR Green/ROX FAST master mix (Qiagen) using the primers listed in Table S4. Relative quantification was calculated as 2(^−ΔΔCT) using β-actin as a reference gene. For analysis of Gdf11 expression from the MACS-purified CD19+ and CD19- splenic cells, real-time PCR analysis was performed with Taqman probes for Gdf11 (Mm01159973_m1, Taqman Gene Expression Assays, Thermo Fisher) and Hprt (Mm01545399_m1, Taqman Gene Expression Assays, Thermo Fisher). Relative quantification was calculated as 2(^−ΔΔCT) using Hprt as a reference gene.

For this purpose, total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufac-turer’s protocol. One microgram of total RNA was reverse-transcribed using Cloned AMV First-Strand cDNA synthesis kit (Invitrogen) in a 20-μl total reaction volume, followed by quantitative PCR on ABI 7500 Fast Thermocycler with RT²SYBR Green ROX Fast Master Mix (Qiagen) using gene specific primers (see Table 1 for primer sequences). For each exponential amplification, PCR was performed for 1 cycle at 95°C followed by 40 cycles at 95°C and 60°C. To verify amplification specificity, melting curves of the PCR products of each primer set were analyzed. Each experiment was prepared in triplicate, and data are represented as means ± S.D. of at least three independent experiments. To normalize for sample variation, expression of glyceraldehyde-3-phosphate dehydrogenase was determined as an internal control.

The Cow Inflammatory Cytokines & Receptors RT² Profiler™ PCR Array (Qiagen) was used for the quantitative real-time RT-PCR (RT-qPCR) analysis of the ICLN of all sheep. Total RNA was extracted as described above and each sample of RNA was reverse transcribed using a RT² First Strand Kit according to the manufacturer’s protocol (Qiagen). Real-time PCR was performed on each cDNA sample using the RT² Profiler™ PCR Array with the RT² SYBR Green/ROX FAST Mastermix in a Rotor-Gene Q cycler (Qiagen). The cycling profile was performed at 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Melting curve analysis of qPCR products confirmed the absence of secondary product. RT² Profiler PCR Array Data Analysis version 3.5 software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) was used for data analysis. Spearman rank-order correlation analysis was carried out using GraphPad Prism 6.07 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).