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Iqbal et al. Vessel Plus 2019;3:40 I http://dx.doi.org/10.20517/2574-1209.2019.28 Page 3 of 13

Figure 1. Effect of RORγ gene deletion on body and liver weights in mice. RORγ WT and KO mice (n = 3) were identified by genotyping
the DNA isolated from tail tissues (A). Total body (B) and liver (C) weights of 8-week old chow diet fed WT and KO mice were measured.
Values were plotted as mean ± SD. P values were calculated using two-tailed Student’s t test. **P < 0.01. WT: wild type; KO: knockout;
RORγ: retinoic acid-related orphan receptor γ

Life Technologies (Carlsbad, CA). Omniscript RT kit (catalog #205113) was from Qiagen (Germantown,
TM
MD). qPCR core kit for SYBR Green I (catalog #10-SN10-05) was purchased from Eurogentec (San
Diego, CA). All other chemicals and solvents were obtained from Fisher Scientific through its local
distributor in the Kingdom of Saudi Arabia.

Animals
Heterozygous RORγ mice were intercrossed to obtain Rorγ -/- and wildtype (WT) littermate
[20]
controls as described previously . Genotyping was performed by PCR analysis of tail DNA using
5’-TGGTAGTGTCACTATCTGTGTCCC-3’ forward and 5’-CTTCAGAACTTATGTCAGGAACTCC-3’
reverse primers to generate an 850-bp WT product and a 250-bp KO product to identify the WT and Rorγ
KO mice [Figure 1A]. For this study, we used eight-week-old chow-diet-fed WT and Rorγ KO male mice
(n = 3) on C57BL/6J background. All experiments were approved by Institutional Animal Care and Use
Committee at King Faisal Specialist Hospital and Research Center.

Body weight and lipid analysis
Mice in WT and Rorγ KO groups were fasted overnight for 18 h, weighed using digital balance to measure
total body weight and sacrificed to collect blood and livers. Liver weight of each animal was recorded.
Blood was used to isolate plasma after centrifugation of the samples for 15 min at 2500 rpm using a
tabletop centrifuge. Plasma and liver tissues were stored at -80 °C until further analysis. Hepatic lipids from
[21]
WT and KO mice were extracted by Bligh and Dyer method . Total cholesterol and triglyceride levels in
the plasma and liver tissues were measured using commercially available kits from Thermo Scientific as
[22]
described previously .