Superscript first strand kit

At the age of 15 weeks (five days after the last injection), plasma and skeletal muscle from 5-FU treated or vehicle treated SOD1G93A mice, as well as wild-type littermates (n = 8 for all groups), were collected and processed for gene expression analysis. Left quadriceps femoris muscles were dissected and immediately frozen in liquid nitrogen. Each muscle was pulverized in liquid nitrogen using a Cellcrusher cryogenic tissue pulverizer (Cellcrusher, Cork, Ireland) and half of the power was kept at -80°C for protein extraction (below). The powered muscle tissue was further homogenized using a PRO200 homogenizer (PRO Scientific Inc) and RNA was extracted with TRIzol reagent (Invitrogen). Potential residual genomic DNA was eliminated using the Turbo DNA-free Kit (Ambion) and 1μg of DNAse-treated RNA was retrotranscripted using the Superscript First Strand kit (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed from 1:10 diluted cDNA in triplicates using StepOne Plus Real-Time PCR System (Applied Biosystems). The gene-specific TaqMan probes (Applied Biosystems) used are indicated in Table 1. Geometric mean of the reference genes Gapdh and Actb (β-actin) was used for normalization [15 (link)] and relative gene expression was determined using the 2^-ΔΔCT method [16 (link)].

Total RNA was purified using Trizol (Thermo Fisher Scientific #15596026), and cDNA synthesized by the SuperScript First-Strand kit (Invitrogen #11904-018). Quantitative PCR was performed by C1000 Touch thermal cycler with the CFX384 optical reaction module (Bio-rad). The expression level of a gene was normalized to that of Gapdh. Each PCR reaction contained 2 µL of cDNA, 5 µL of the iTaq Universal SYBR Green Supermix (Bio-rad #1725124), and 500 nM of forward and reverse primers. The primer sequences are shown in Table 1.Table 1

Primer sequences for RT-qPCR.

Myrf	Forward	CATTGTGCGGGCCTCTAACCC
	Reverse	CCTCATCTGGCCGGTCGG
Dagla	Forward	GCCGCACCTTCGTCAAGC
	Reverse	GACCAGCTGGTGGCCTGAC
Syt7	Forward	CCACTGGTGTCAGCGCAAACTG
	Reverse	GCTTTCTTCTCACCGCGCCC
Sdhaf2	Forward	CCTTGATCCCGACGCTGGC
	Reverse	GAGTCTGTTGGGCTGTCACCTCTG
Cpsf7	Forward	CCCAAGAGGGGGAATACCTCCAC
	Reverse	GGGCTTATCCACACGAGCAGATGAG
Tmem258	Forward	CCTGGTTCTTCGTTTACGAGGTCAC
	Reverse	GGAGGAAGAGGACTCCAAAGCC
Fads1	Forward	CCCCTCTTCTTCGCCCTG
	Reverse	GGGGTCCGATGAGGAAGAAGTAC
Gapdh	Forward	GGTGAAGGTCGGTGTGAACGG
	Reverse	CTGGAACATGTAGACCATGTAGTTGAGG

The Trizol reagent (Invitrogen) was used to purify the total RNA from the cells. After synthesis of the first-strand cDNA using Superscript First-Strand Kit (Invitrogen), the PUMA and GAPDH mRNA levels were determined by qPCR using primers purchased from Integrated DNA Technologies. The relative PUMA mRNA levels were calculated by the 2−ΔΔCt method with three replicates (Livak and Schmittgen, 2001 (link)).

For sequencing of ENAH from Met4 cells, 1 μg of total RNA was used to synthesise cDNA using a SuperScript First-Strand kit (Invitrogen). cDNA (Supplementary Table 1) was amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs) in PCR reactions consisting of an initial denaturation step of 30 s at 95 °C, followed by 30 cycles at a denaturation temperature of 95 °C (30 s per cycle), an annealing temperature of 60 °C (30 s per cycle) and an extension temperature of 72 °C (1 min per cycle). A final extension step was performed at 72 °C (10 min). PCR product was analysed on a 1% (w/v) agarose gel, excised and purified using a gel extraction kit (Qiagen). PCR product was cloned into pCR-Blunt II-TOPO (Invitrogen) by blunt-end ligation according to manufacturer’s instructions and transformed into DH5α cells. Plasmid was isolated using a QIAprep Miniprep kit (Qiagen) and sequenced using M13 sequencing primers (Supplementary Table 1).

Cells were seeded to 70% confluency in 10 cm dishes in complete growth media. Cells were collected in Trizol (Invitrogen) and RNA was isolated according to manufacturer's instructions. The SuperScript First-Strand kit (Invitrogen) was used for cDNA synthesis. RT² SYBR Green Flour FAST Mastermix (Qiagen; Valencia, CA) was used in qPCR reactions. Cycling conditions were 95°C 10 minutes, followed by 40 cycles of 95°C 10 seconds and 55°C 30 seconds. Melt curves were assessed for each run and reverse transcriptase negative and template negative controls were used. Reactions were carried out using a Bio-Rad CFX96 Real-Time PCR detection System with Bio-Rad CFX Manager 3.0 software. Primers were designed using Integrated DNA Technologies (Coralville, IA) PrimerQuest software and sequences used include (5′ to 3′): ACACA forward: CTTGTCACCTGCTTCTGT, ACACA reverse: AGACATGCTGGACCTTATG; ACLY forward: TTCGGCAGAGACAGGTA, ACLY reverse: GAGGTGGTACAGATGAACTT; GAPDH forward: GTCGGAGTCAACGGATTT, GAPDH reverse: AGTTGAGGTCAATGAAGGG.

Extraction of total RNA from untreated and treated THP1 cells (3 × 10⁶) was performed using the Illustra RNAspin Mini Isolation Kit (GE Healthcare, Little Chalfont, UK). Total RNA was used to monitor the mRNA expression of human TLR4. Complementary DNA (cDNA) was produced from total RNA extracts using the SuperScript first-strand kit (Invitrogen – Life Technologies, Carlsbad, CA, USA) and performed in a Tetrad2 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Primer pairs (Invitrogen) and the sequences used were: 5′-GAA GCT GGT GGC TGT GGA-3′ (sense) and 5′-TGA TGT AGA ACC CGC AAG-3′ (antisense) for human TLR4, as previously described [33 (link)]. The β-actin primer sequences used were 5′-TGA TGA CAT CAA GAA GGT GGT GAA G-3′ (sense) and 5′-TCC TTG GAG GCC ATG TGG GCC AT-3′ (antisense). Real-time PCR was done using the QuantiTect Reverse Transcription kit containing PCR SyberGreen Master Mix (Qiagen, Hilden, Germany) to quantitatively monitor the mRNA expression of TLR4 compared with β-actin gene expression (a house-keeping gene used as an internal control). For each analysis, a negative control was prepared using all the reagents except the cDNA template. All the reactions were run in triplicate.