Taqman kits

RNA was purified from MGEC and MMEC using the RNeasy micro-kit (Qiagen, Hilden, Germany) and reverse-transcribed to complementary DNA (cDNA) using the first-strand cDNA synthesis kit (Thermo Fisher Scientific). The mRNA levels of PPAR β/δ, ANGPTL4, elastin, collagen 3α, fibronectin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were assessed in triplicate by real-time PCR using TaqMan kits (Applied Biosystems (Waltham, MA, USA) assay IDs: Hs00987008_m1, Hs00211522_m1, Hs00355783_m1, Hs00915125_m1, Hs01549976_m1 and Hs03929097_g1) and the StepOne real-time PCR system (Applied Biosystems). The comparative Ct method, with GAPDH as the reference gene, and the 2^−ΔΔCt formula were used for the relative quantification of mRNA levels.

Total RNA was extracted from cells (5 × 10⁵) using RNeasy Mini kits (Qiagen, Valencia, CA) and reverse transcribed with TaqMan kits (Applied Biosystems, Foster City, CA). Quantitative PCR was performed in triplicate using SYBR select master mix (Applied Biosystems) on a 7300 or 7500 real-time PCR system (Applied Biosystems). Specificity of amplification was confirmed by melt curve analysis. Cycle threshold (C ) values for target isoforms were normalized to the indicated endogenous reference gene. Primer sequences (Supplemental Table 1) were designed from UCSC genome browser reference transcript sequences using Primer3.

RNA was isolated from the fresh brain tissue, using the commercially available extraction kit RNeasy^® Blood/Tissue kit (Qiagen, Germany) according to the manufacturer’s instructions. RNA quantification and purity were evaluated using a Nanodrop Spectrophotometer (Thermo Scientific). Samples with an absorbance ratio at 260/280 nm between 1.5 and 2.0 were considered acceptable. RNA degradation was not assessed but according to previous reports, it remains stable for at least 36 h post-mortem (White et al., 2018 (link)). cDNA was synthesised with an available commercial kit (Nzy First-Strand cDNA Sunthesis Kit) in a BiometraAlfagene thermocycler, according to the manufacturer’s instructions. P2X7R (hs0017521_m1) and the reference gene Ubiquitin C (UBC; hs00824723_m1) expression were quantified by Real Time PCR with specific primers and probes (Taqman^® Kits, Applied Biosystems, USA) and a NzySpeedy qPCR mastermix (Nzytech, Portugal) in Corbett Rotor Gene 600 Real Time Thermocycler machine (Corbett Research, UK). UBC gene was chosen as the reference gene since its expression showed relatively low variability in expression levels in the regions studied (Trabzuni et al., 2011 (link)). Each reaction was performed in triplicate and the average Ct value was used in the analysis. The relative expression was calculated using the 2^−ΔΔCT method.

Total RNA from cell, tissue or plasma was reversed transcribed for qRT-PCR analysis. The expression of a panel of miRNAs, and the mRNA expression of p62 and NQO1, were assessed using TaqMan kits (Applied Biosystems) according to procedures described previously [1 (link), 9 (link)]. RNU6B, miR-16 and GAPDH were used as the internal controls. The threshold cycle (Ct) method was used to measure the relative changes in expression. The −ΔΔCt was the difference of ΔCt values between different experimental settings and the sample groups. The 2^−ΔΔCt indicates fold of change in expression.