Cfx384 light cycler

Spinal cord, brain and quadriceps muscle samples were pulverised under liquid nitrogen and the powder mixed with TRIzol reagent at a ratio of 1ml TRIzol per 100mg tissue (quadriceps muscle) or 1ml per 50mg (brain and spinal cord). Samples were extracted from tissue samples using a TRIzol protocol as supplied by the manufacturers with the addition of an extra chloroform extraction step (1:1) after collection of the aqueous phase. RNA was assessed via nanodrop to ensure good 260/280 and 260/230 ratios (>1.9). Samples with 260/230 ratios lower than 1.9 were cleaned by a second round of isopropanol precipitation.
For cDNA synthesis, 800ng of RNA per reaction was reverse transcribed using the RTnanoscript2 kit (Primer Design) and final reactions were diluted 1/5 (empirically determined to be a sufficient dilution to prevent cDNA synthesis reagents having any effect on subsequent PCR efficiency). qPCR was performed in a CFX384 light cycler (Biorad) using PrecisionPLUS mastermix with SYBR green (Primer Design), with 10ul volumes of primers at a final concentration of 500nM, and with 10-15ng cDNA per well. Cycling conditions: 2mins at 95°C, then 50 cycles of: 15sec 95 ^oC, 20sec 60°C, 20sec 72 ^oC followed by a melt curve (60–95 ^oC in 0.5 degree steps).
The following primers were used:
Pak1ip1 was used as a reference gene in all tissues (Primer Design).

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)).