Abi taqman universal master mix

Putative causative mutations in MC1R and ASIP were further analyzed for genotype–phenotype association by Sanger sequencing the regions in 69 dromedaries (29 white, 17 black, 23 brown). Custom TaqMan^TM SNP genotyping assays were designed for MC1R and ASIP mutations according to manufacturer specification (Applied Biosystems) (Table 2), and used for genotyping all 188 dromedaries. We used CFX-96 Real Time-PCR machine (Bio-Rad) and corresponding software for PCR amplifications, genotyping and allelic discrimination. The thermal conditions were: priming at 60°C for 1 min, initial denaturation at 95°C for 10 min, 40 cycles of 92°C for 15 s, annealing at primer-specific t°C, extension for 1 min at 60°C, followed by a final extension at 65°C. The 8 μL reactions contained 0.208 μL of TaqMan^TM assay, 30 ng template DNA and 4.2 μL of ABI TaqMan Universal Master mix, no UNG (Applied Biosystems).

Total RNA was isolated from frozen IWAT and EWAT samples, as previously described. Briefly, frozen tissue was homogenized in Trizol reagent and phase separation was carried out using chlorofom per the manufacturer’s protocol (Sigma Aldrich, St. Louis, MO, USA). 70% ethnaol was added to the aqueous layer, which was then further purified using a RNeasy mini kit (Qiagen, Valencia, CA, USA) per the manufacturer’s protocol. RNA quantity and quality were analyzed by spectrophotometry (NanoDrop, ND-1000, Thermo Scientific, Wilmington, DE, USA), and a cDNA library was created using M-MLV reverse transcriptase (Promega, Madison, WI, USA) per the manufacturer’s protocol.
qRT-PCR was performed using commercially available Applied Biosystems Taqman primers and probes (Cd11b, Mm01271255_m1; Cd68, Mm04411920_m1; F4/80, Mm00802527_m1; Mcp-1, Mm00441242_m1; Applied Biosystems, Foster City, CA, USA) and samples were run in duplicate on the ABI 7900HT platform (Applied Biosystems, Foster City, CA, USA) using ABI Taqman Universal MasterMix (Applied Biosystems, Foster City, CA, USA). Gene expression was analyzed using a standard curve and normalization to cyclophilin as the endogenous control. Data are expressed as arbitrary units (AU).

Previously published TaqMan™ SNP genotyping assays for the DMRT3 lateral gait associated allele were utilized for gait analysis [35 (link)] in the ENH and the 8 related breeds (Group A). Of the latter, 6 breeds (Finn Horse, Konik, Gotland Pony, Icelandic Horse, Norwegian Fjord, and Shetland Pony) have already been genotyped for the DMRT3 gait allele [36 (link)]. Therefore, we genotyped the remaining 3 breeds for the gait mutation in a total of 114 horses: 33 ENH, 52 Hucul Horses, and 29 Exmoor Ponies. We used a CFX-96 Real Time-PCR machine (Bio Rad, Hercules, CA, USA) and corresponding software for PCR amplifications, genotyping and allelic discrimination. The thermal conditions were: priming at 60 °C for 1 min, initial denaturation at 95 °C for 10 min, 40 cycles at 92 °C for 15 sec each, annealing at 60 °C for 30 s, and extension at 60 °C for 1 min, followed by a final extension at 65 °C for 10 min. The 8 µL reactions contained 0.208 µL of TaqMan™ assay, 30 ng template DNA and 4.2 µL of ABI TaqMan Universal Master mix, no uracil-N-glycosylase (UNG) (Applied Biosystems).