RNA extraction, CDNA synthesis and quantitative PCR

RNA was extracted using Tri-reagent (Sigma-Aldrich), purified using the RNeasy mini kit (Qiagen) and treated with Ambion DNA-free (Life Technologies). cDNA was made from 1 μg of RNA using the sensiFAST cDNA synthesis kit (Bioline, London, UK). 2 μl aliquots of cDNA were used in qPCR. Primers were designed using NCBI Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primer sequences can be found in Table 1. Quantitative PCR (qPCR) was carried out using SYBR Green containing supermix (Bioline) on the Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad), and all PCR reactions performed in duplicate. PCR cycle parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 15 s. At the end of the program a melt curve was produced by taking readings every 1°C from 65°C to 95°C. The levels of 18S rRNA did not change between treatments (data not shown) and it was used to normalise gene expression using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). An unpaired Student's t-test was used to determine significant differences in the mean gene expression using iPSCs from three independent horses versus three lines of iPSCs derived from the same horse.

Primer sequences for equine gene transcripts