Precisionplus sybr green mastermix

qPCR reactions were performed in triplicate with 2 μl cDNA per well (∼8 ng), using PrecisionPLUS SYBR Green Mastermix (PrimerDesign). Primers to SDHA, UBC and YWHAZ [previously validated reference genes for canine brain (Crawford et al., 2021 (link))] were taken from the C. familiaris geNorm kits (PrimerDesign) and are proprietary. qPCR primers to canine dystrophin were used as previously published (Hildyard et al., 2020a (link)). PCR was conducted in a CFX384 light cycler (Bio-Rad) in a three-step PCR (95°C, 15 s; 60°C, 20 s; 72°C, 20 s for 40 cycles) with subsequent melt curves performed for all reactions. All primer pairs gave sharp, single-amplicon products and single melt peaks. Quantification cycle (Cq) values were determined by regression. Relative quantities (RQ) were calculated for the full data set by using the minimal Cq value across all isoforms to enable assessment of individual isoform expression relative to total dystrophin expression (sum of expression of all isoforms). RQ data were then normalised to the geometric mean of three previously validated reference genes: SDHA, UBC and YWHAZ (Crawford et al., 2021 (link)).

RNA isolation from macrophage in culture was performed using the RNeasy UCP kit (Qiagen) according to manufacturer’s instructions. cDNA was transcribed from the total RNA using the Precision nanoScriptTM 2 RT kit (Primerdesign Ltd.) according to the manufacturer’s instructions. Real time qPCR was performed using a Bio-Rad i-Cycler. The reagents used were Precision PLUS SYBR-Green master mix (Primerdesign) and specific primers (Supplementary Table 1) designed with NCBI BLAST. All assays were performed in triplicate and normalised to the expression levels of GAPDH, determined in our assays as the most suitable house-keeping gene in Trib3 deficient macrophages (Supplementary Figure 1).

The 19 shoot samples were used for qPCR to verify RNA-Seq gene expression calls using primers for four target genes and primers for 18S [74 (link)] as an endogenous control. cDNA and a reverse transcription (RT) control were produced using a QuantiTect Reverse Transcription Kit (QIAGEN), incorporating a DNA removal step. qPCR reactions, no template controls and RT controls, were conducted in triplicate using 10 μl PrecisionPlus SYBRgreen Mastermix (Primerdesign, UK), 200 nM per primer and 1 μl cDNA or deionized water in a 20 μl reaction. Reactions were conducted using a realplex Mastercycler epgradient S (Eppendorf, Germany), and standard curve data was used to calculate reaction efficiencies for all primer pairs. Melt curves were employed to check for non-specific amplification and contamination. Expression was normalized to 18S, and statistical analyses were conducted using GLMs and post hoc Tukey tests in Minitab. Where there was non-normality, log₂-transformed data was used. Pair-wise fold changes and standard errors plus log₂FCs were calculated from the mean normalized expression levels for each treatment, and regressions of RNA-Seq log₂FC against qRT-PCR log₂FC were conducted in SigmaPlot 2001.