Eco software v4

TRIZOL® (Life Technologies, Grand Island, NY) was used to isolate total RNA from 50 mg of each frozen liver tissues by following the manufacturer’s protocol. The isolated RNA concentration was measured by Nanodrop® ND-1000 (Thermo Scientific, Wilmington, DE). Real-time PCR amplification reactions (in a final volume of 20 μL) were carried out in an Eco-Real-Time PCR system from Illumina (San Diego, CA) by using Power SYBR® Green RNA-to-CT™ 1-step kit from Life Technologies (Grand Island, NY) according to the manufacturer’s protocols. For the PCR reactions, specific forward and reverse primers (200 nM) and 50 ng for each target RNA were used. A dissociation curve was generated to distinguish the specific amplicons from non-specific amplicons. The Ct-values were calculated with using Eco® software V4.0 (Illumina). The specific primers (shown in Table 1) to analyze the mRNA levels for TLR4, CCR2, leptin, leptin receptor, MCP-1 and beta-actin, respectively, were designed using Primer-BLAST software.

This study was conducted according to the MIQE guidelines (Bustin et al., 2009 (link)). cDNA was analyzed by quantitative PCR using FastStart Essential DNA Probes Master (Roche) according to the manufacturer’s protocol. Thermal cycling was conducted using the Eco machine (Illumina, San Diego, CA, USA) and the same conditions were used for all target genes: 95 °C for 10 min for polymerase activation, followed by 45 cycles of 95 °C for 10 s and 60 °C for 30 s for denaturation and annealing/elongation, respectively. All reactions including plate controls and blank controls were run in triplicate. Plate controls include identical reaction materials on every run. A stable quantification cycle (Cq) value from all plate controls allowed data from multiple plates to be consolidated into a single data set. Threshold value for each candidate gene was manually set (Table 2). Baseline values were assigned for all plates using the Eco Software V4.0 (Illumina). PCR amplification efficiency (E) was calculated as E = (10^{(−1∕slope)} − 1) × 100%, where slope is the gradient of a standard curve. A gene-specific E for the following normalized value (NV) calculation was obtained from the average of at least three E values for each gene.

To determine the tolerance of commercially available RT and PCR enzymes to blood, one-step reaction master mixes were assembled on wet ice using GoTaq® G2 Hot Start polymerase (Promega; Southampton, UK) in GoTaq® G2 Hot Start Colorless Master Mix (Promega) supplemented with 5 units of GoScript® Moloney Murine Leukemia Virus reverse transcriptase (Promega), primers and probes/dyes at concentrations as previously published.6 (link),27 Duplicate aliquots were dispensed, and supplemented with blood and nuclease-free water (Promega) to achieve the final blood concentrations indicated. Template was added last in each aliquot to achieve a 50 μl final reaction volume ahead of commencing thermal cycling on an Illumina Eco™ running Eco™ Software v.4.0.7.0 (Illumina Inc., San Diego, CA, USA).