Rotor gene q series software 1

Total RNA was isolated from muscle biopsies using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany; including Dnase I treatment) and subsequently the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions. A Micro-Dismembrator (B. Braun Melsungen AG, Melsungen, Germany) was used for tissue disruption and homogenization. A cDNA synthesis with integrated removal of genomic DNA contamination was performed with the QuantiTect Reverse Transcription Kit (Qiagen).
Analysis by qPCR was performed on a Rotor-Gene 2000 real-time PCR thermocycler (Qiagen, Hilden, Germany; program: 40 cycles of 95°C for 10 s followed by 60°C for 45 s) using Power SYBR Green Supermix (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions. Data were quantified using Rotor-Gene Q Series Software 1.7 (Qiagen). Efficiencies were calculated from the slope of template dilution curves with primers for genes of interest (MHC isoforms or genes of energy metabolism) and the reference gene (BSM or RPS12, respectively), and used for quantification of changes of transcript levels by the ΔΔCt-method (Livak and Schmittgen, 2001 (link)). The efficiency of all primer sets was between 95 and 105%.

Eight DEGs that were significantly differentially expressed in at least one isolate-infected group in each tissue were selected for the quantitative reverse transcript PCR (RT-qPCR) validation. As mentioned above, total RNA was extracted from spleen and liver tissue samples. The manufacturer’s instructions were followed to produce cDNA from the total RNA using a PrimeScript RT reagent Perfect Real Time Kit (TaKaRa, Dalian, China). Then, the reaction of qPCR was performed and analysed using a Rotor-Gene Q Series Software 1.7 supplied with the instrument (QIAGEN, Hilden, German). An amount of 10 μL of TB Green Premix Ex Taq Ⅱ (TaKaRa, Dalian, China), 2 μL of the cDNA sample, 0.8 μL (10 μM) of each primer, and ddH2O in a total volume of 20 μL made up the reaction mixtures. The reactions were amplified for 30 s at 95 °C, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s.
Primer sequences of eight DEGs were listed in Table 1. For normalization of gene expression, β-actin gene was used as an internal control. Primers had a Tm of roughly 60 °C, and PCR products ranged in length from 100 to 200 bp. qPCR was conducted three times for each sample as technique replicates.