Genorm kit

Changes in the relative gene expression were analysed by RT-PCR. The EA.hy926 cells were cultured in 6-well plates (Corning, Corning, NY, USA) (cell density of 6 × 10⁵ cells/mL) with FAs for 48 h and then without FAs for 6 h. Taqman Gene Expression Primers (Thermo Fisher Scientific, Waltham, MA, USA) were used to determine the expression of NFκB1 (Hs00765730_m1), NFκBIA (Hs00355671_g1), IκBKB (Hs00233287_m1), PPARα (Hs00947536_m1), PTGS2 (Hs00153133_m1) and IL-6 (HS00985639_m1). Total RNA was extracted from the cells using the ReliaPrep RNA cell Miniprep System (Promega, Southampton, UK). RNA quantity and quality were analysed by NanoDrop. RNA integrity was assessed as RIN score using an Agilent Bioanalyzer (RNA Total Eukaryote 2100 Nano). cDNA was synthesised from total RNA using GoScript Reverse Transcriptase (Promega, Southampton, UK). Housekeeping reference genes were determined using a geNorm Kit (Primerdesign, Camberley, UK): YWHAZ (Hs01122445_g1) and RPL13A (Hs04194366_g1) were used as housekeeping genes to determine quantitative gene expression.

Total RNA was extracted from 15 NB cell lines and 76 NB samples using QIAzol Lysis Reagent (Qiagen) according to the manufacturer's instructions, and reverse transcription performed with iScript Reverse Transcription Supemix kit (Biorad). RNA quality was checked by 2100 BioAnalyzer using RNA 6000 Nano LabChip kit (Agilent Technologies) and quantified by NanoDrop (Thermo Scientific). Only RNAs with a RIN > 7 were included in subsequent experiments. The geNORM kit (Primerdesign) was employed to select the most stable housekeeping genes in our experimental set of samples that resulted to be 18S-RNA and GAPDH in NB cell lines and 18S-RNA, GAPDH and UBC in NB patients. RT-qPCR was carried out on the Mastercycler ep Realplex S (Eppendorf) using TaqMan FAM-labeled probes for GALNT14 (Assay ID: Hs00226180_m1, Life Technologies) and for 18S-RNA, GAPDH and UBC (Primerdesign).

Cellular total RNA was isolated using GeneJET RNA purification kit (Life Technologies) according to the manufacturer's protocol. First-strand cDNA was synthesized using the nanoScript reverse transcription kit (Primer Design, Southampton, UK). Quantitative PCR (qPCR) amplification was performed in 96-well plates in a mastermix for probes (Roche, Burgess Hill, UK) and run on a LightCycler^® 96 System (Roche). The qPCR amplifications for the human AXL (assay I.D. Hs.PT.56a.1942285), MERTK (assay I.D. Hs.PT.58.2640315), TYRO3 (assay I.D. Hs.PT.58.38778546) and GAS6 (assay I.D. Hs.PT.58.21535693) genes were performed using pre-designed primers/probes purchased from Integrated DNA Technologies (Leuven, Belgium). The amplification procedure entailed 45 cycles of 95°C for 10sec followed by 60°C for 30sec. Relative expression analysis was performed using the equation N = N₀ x 2^Cp (LightCycler®96 software; Roche), normalising against the gene for ATP synthase subunit beta (ATP5B), which was determined in-house as the best internal reference gene out of 12 genes tested (geNorm kit, Primerdesign, Southampton, UK; data not shown).

Total RNA was extracted using the RNeasy mini kit according to the manufacturer’s instructions, which includes a DNase I treatment to remove genomic DNA (Qiagen). RNA was quantified using a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific) and stored at − 80 °C. Using the MMLV Superscript reverse transcriptase (Invitrogen) and random hexamers (Operon), reverse transcription (RT) was performed as described previously^{48 (link)}. Taqman quantitative real-time PCR (qPCR) was performed on the using the CFX96 C1000 real-time thermal cycler (Bio-Rad). The levels of gene expression were determined by normalizing to housekeeping genes, and the relative levels of the transcripts were determined by using the 2^−ΔΔCt method^{48 (link),49 (link)}. The expression levels of a ‘gene of interest’ was normalised to the geometric mean of endogenous housekeeping genes. The reference genes were selected from a panel based on their stability in R6/2 and WT samples using the Genorm kit (Primer Design) and were Hprt, Canx, and Eif4a2 for liver and Hprt, B2m and Atp5b for spleen. We did not have sufficient RNA to determine the stability of a panel of reference genes for the peripheral macrophages and therefore, these reference genes were based on previous data^{16 (link)}. Primer sequences and probes used are summarized in Supplementary Table S5 online.

cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). SYBR Green reagents (Thermo Fisher) were used for qPCR analysis, and primers were used at 40 μM concentration (Supplementary Table 4). mRNA concentrations were normalized against two commonly used “housekeeping” genes: GAPDH and ATP5B (determined by geNorm kit, PrimerDesign).
Tissue samples were grouped according to age and region of colon. Age groups were defined as “adults” (25–60 years) and “elderly” (≥70 years) and ascending colon and descending colon were examined separately; adult ascending n = 9, elderly ascending n = 10, adult descending n = 10, elderly descending n = 10. These groups were taken from previously published research which defined an age- and region-dependent decline in neuromuscular function of human colon (Broad et al., 2019 (link)). The adult samples had an age range of 27–60 years (median 53) and were 53% male. The elderly samples had an age range of 70–89 years (median 79) and were 50% male.