Lightcycler480 sybr green master 1 kit

Primary culture of grass carp hepatocytes was prepared by collagenase digestion (41 (link)) and maintained in 24-well plates at a seeding density of ~0.7 × 10⁶ cells/ml/well. After drug treatment, total RNA was extracted from individual wells by TRIZOL, digested with DNase I, and reversely transcribed using Superscripts II (Invitrogen) with Oligo-dT as the primer. RT samples prepared were then subjected to real-time PCR for TNFα and type II SOCS mRNA measurement using a LightCycler 480 SYBR Green Master I Kit (Roche) with the RotorGene-Q qPCR System (Qiagen). In parallel experiments to evaluate GH resistance at the hepatic level, carp hepatocytes were exposed to GH with co-treatment of LPS or TNFα and the RT samples prepared were used for real-time PCR measurement of GHR and IGF-I/-II transcripts. Real-time PCR for respective gene targets, including TNFα, GHR, IGF-I/-II, and different members of type II SOCS, will be conducted according to the conditions described in Supplemental Table 1. In our studies, serial dilutions of plasmids with the ORF of the target genes were used as the qPCR standards for data calibration and parallel measurement of 18S RNA expression was used as the internal control. After the assays, the authenticity of PCR products was routinely confirmed by melting curve analysis.

Total RNA from cultured cells was extracted using RNeasy kit (QIAgen, UK), while RNA from murine muscles (as described in the animal study below) was extracted using RNeasy Fibrous Tissue kit (QIAgen, UK), following the manufacturer’s instructions. Tissue homogenization was performed in the lysis buffer provided with the kit, at 25 Hz for 2–4 min, on a TissueLyser II (QIAgen, UK). RNA was quantified on an ND-1000 NanoDrop spectrophotometer (Thermo Scientific, UK). One microgram RNA was reverse transcribed using QuantiTect reverse transcription kit (QIAgen, UK). Ten nanograms of diluted cDNA in qPCR water (Roche, UK) were then amplified using LightCycler480 SYBR Green Master I kit (Roche, UK), according to the manufacturer’s instructions; samples were prepared in triplicates. Reactions were run on LightCycler480 System, initialized at 95°C for 5 min, followed by 45 cycles at 95°C for 15 s, 58^o–62°C for 15 s and 72°C for 15 s. Relative quantification for DUX4 or its relevant genes was performed against corresponding housekeeping genes, B2M or Gapdh. Primers were purchased from Integrated DNA Technologies (Belgium) and are detailed in Supplementary Material, Table S3.

The total RNA from liquid-nitrogen-snap-frozen muscles was isolated using RNeasy Fibrous Tissue kit (QIAgen, Manchester, UK) by following the manufacturer’s instructions. The samples were homogenized in the lysis buffer provided with the kit on a TissueLyser II (QIAgen, Manchester, UK) at 25 Hz for 2–4 min. One microgram of RNA was reverse transcribed using a QuantiTect reverse transcription kit (QIAgen, Manchester, UK). Ten nanograms of diluted cDNA in qPCR water (Roche, Burgess Hill, UK) were then amplified using a LightCycler480 SYBR Green Master I kit (Roche, Burgess Hill, UK) according to the manufacturer’s instructions, with each sample prepared in triplicates. qPCR reactions were run on LightCycler480 System, initialized at 95 °C for 5 min and followed by 45 cycles at 95 °C for 15 s, 58–60 °C for 15 s, and 72 °C for 15 s. The relative quantification for DUX4 and DUX4-related genes was performed against the corresponding housekeeping gene, Gapdh, using Pfaffl’s method as described in [40 (link)]. Data are shown as fold-changes compared to the SCR values obtained in the same way. The primers were purchased from Integrated DNA Technologies (Belgium) and are detailed in Table 1.