Six genes reported as adipocyte specific within the literature and a gene previously validated as a stable reference gene (REF) were analyzed using RefFinder to select the most stable REF to investigate the ability of cell specific genes to standardize relative gene expression across tissues varying in cell type heterogeneity. Angus × Hereford steers (n = 16) were randomly selected and samples of subcutaneous (s.c.), mesenteric (MES), and longissimus dorsi (LM) fat depots were collected, flash-frozen, and stored at −80°C for subsequent analysis. Lipogenic relative gene expression values were either unstandardized (RAW), or standardized with the most stable REF (Thy-1 cell surface antigen; Thy1), second most stable (glyceraldehyde 3-phosphate dehydrogenase; GAPDH), or the geometric mean of GAPDH and Thy1 (GM). Fold changes are presented in Table 1 for tissues differing in heterogeneity (high fat, MES vs. high muscle, LM). Acetyl CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl CoA desaturase (SCD) were all affected by tissue (P < 0.05). Standardization method and the interaction of method and tissue did not alter fold change (P > 0.10). This may suggest that the use of an adipocyte-specific REF does not account for variable cell type heterogeneity. More work is warranted to identify an effective method to standardize qPCR results to tissue heterogeneity, allowing for objective across tissue comparisons. Table 1. The effects of standardization and tissue on the average fold change of relative gene expression

Method1	Tissue2	Gene
ACC	FASN	SCD
GAPDH	MES	0.65	−3.46	−2.63
GM	MES	0.09	−3.51	−2.68
Thy1	MES	−0.01	−3.61	−2.79
RAW	MES	−0.25	−5.07	−3.88
GAPDH	LM	2.04	2.89	1.73
GM	LM	1.77	2.62	1.53
Thy1	LM	1.52	2.42	1.35
RAW	LM	1.78	3.12	1.68
	SEM3	1.826	7.284	6.979
P-value4	T	<0.01	0.02	0.04
M	0.10	0.29	0.33
T × M	0.11	0.35	0.42

¹Standardized against GAPDH, Thy1, the geometric mean (GM) of GAPDH and Thy1, or raw values. ²High fat, MES and high muscle, LM tissue. ³Pooled standard error of means. ⁴Main effect of tissue (T), method (M), and the interaction of T and M.