Quantitative real-time PCR: Liver

Three LXR-targeted genes (CYP7A1, PLTP, and TTPA) were selected for further evaluation in liver tissue. Samples included 10 post-mortem confirmed eNAD-affected horses (n=9 classified as severe eNAD/EDM and n=1 classified as moderate eNAD; age range 1–2 years; median 1 year; n=3 females, n=7 males) and 9 unaffected horses (age range 0.5–13 years; median 2 years; n=6 females, n=3 males) (Table A.1). Age was not significantly different between groups (Mann-Whitney test; P=0.18). The 5 eNAD-affected and 5 unaffected horses used for RNA-seq of the spinal cord in this study were included in these samples. Primers for the genes of interest and 2 previously validated reference genes (ACTB, HPRT1) (6) were designed as described above (Table A.3). Reverse transcription, quantitative PCR reactions and data analyses were performed as described above.