Taqman gene expression assay

RNA from adherent splenocytes was isolated using the RNeasy Mini kit (Qiagen) in conjunction with the RNase-Free DNase Set (Qiagen). cDNA was synthesized using the high capacity cDNA reverse transcription kit according to manufacturer's instructions (ThermoFisher). Relative quantitation of mRNA levels was performed by quantitative PCR via TaqMan Gene Expression Assays (ThermoFisher) and TaqMan Gene Expression Master Mix (ThermoFisher) using a StepOnePlus and a 7500 Fast System (ThermoFisher). Analyses were performed using the standard curve method with β-actin as the normalizing endogenous control. The following TaqMan Gene Expression Assays were used (ThermoFisher): Tlr1 Mm00446095_m1, Tlr2 Mm00442346_m1, Tlr3 Mm01207404_m1, Tlr4 Mm00445274_m1, Tlr5 Mm00546288_s1, Tlr6 Mm02529782_s1, Tlr7 Mm00446590_m1, Tlr8 Mm04209873_m1, Per2 Mm00478113_m1, Nr1d1 (Rev-erbα) Mm00520708_m1, Actb Mm00607939_s1, Tnf Mm00443258_m1, Il6 Mm00446190_m1, Ifnb Mm00439522_s1, Il1b Mm00434228_m1.

RNA from splenocytes was isolated using the RNeasy Mini kit (Qiagen) in conjunction with the RNase-Free DNase Set (Qiagen). cDNA was synthesized using the high capacity cDNA reverse transcription kit according to manufacturer’s instructions (ThermoFisher). Relative quantitation of mRNA levels was performed by quantitative PCR via TaqMan Gene Expression Assays (ThermoFisher) and TaqMan Gene Expression Master Mix (ThermoFisher) using a StepOnePlus (ThermoFisher) system. Analyses were performed using the standard curve method with β-actin as the normalizing endogenous control. Relative quantitation values were determined by dividing the relative quantity for the target gene by the relative quantity for β-actin. The following TaqMan Gene Expression Assays were used (ThermoFisher): Actb Mm00607939_s1, Per2 Mm00478113_m1, Rev-erbα Mm00520708_m1, Bmal1 Mm00500226_m1, Dbp Mm00497539_m1, Tlr1 Mm00446095_m1, Tlr2 Mm00442346_m1, Tlr3 Mm01207404_m1, Tlr5 Mm00546288_s1, Tlr6 Mm02529782_s1.

Frozen spinal cord samples were dissected macroscopically to separate distinct grey and white matter regions. Weighed samples of grey and white matter (~ 10 mg) were then prepared for gene expression analyses using manufacturer’s instructions as follows: Nucleic acids were extracted (TRI-Reagent, Sigma); contaminating gDNA in isolated mRNA was degraded (Turbo DNA-Free Kit, Thermo Fisher Scientific); mRNA quantity was measured (Qubit RNA HS Assay Kit, Thermo Fisher Scientific); cDNA was synthesised (High Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific); and cDNA (25 ng) was pre-amplified for all genes assessed (Taqman PreAmp Master Mix and Taqman Gene Expression Assays (Supplementary Table 1), Thermo Fisher Scientific). Pre-amplified cDNA was diluted 20-fold, then quantitative RT-PCR performed (Taqman Gene Expression Assays and Taqman Fast Advanced Mastermix, Thermo Fisher Scientific) on samples in triplicate using a QuantStudio 6 Flex system (Thermo Fisher Scientific). Relative expression of individual genes involved in cellular copper handling was determined via the ΔΔct method normalised to GAPDH. Composite z-score values representing changes affecting overall copper handling were calculated for each control or ALS case as the average z-score across all genes analysed.

cDNAs were synthetized by reverse transcription using SuperScript III First-Strand Synthesis SuperMix (Life Technologies). Expression of mouse Acta2, Col1a1, Ccl2, Cd68, Il6, Timp1, and Tgf-β1 was analyzed by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific) on Applied Bioscience 7900HT Fast Real-Time PCR system. Expression of human HDC, IL6, IL1β, TNFα, HRH2, and CREB5 was analyzed by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific) on the CFX96 Touch Real-Time PCR Detection System PCR system (Bio-Rad). Expression of human CCL2 and TGF was analyzed using iTaq™ Universal SYBR® Green Supermix (Bio-Rad). The 2^−ΔCT method was applied for relative quantification of mRNA with normalization to either 18S or GAPDH mRNA. The list of the primers is provided in Supplementary Table 4.