Evaluation of reference genes for gene expression studies in human brown adipose tissue

Figures & data

Table 1. Microarray based evaluation and selection of putative reference genes

Gene Title	Gene Symbol*	PR-BAT CV	PT-BAT CV	CV score**
Commonly used reference genes
Beta-2-microglobulin	B2M	0.075	0.111	0.0084
Ubiquitin C	UBC	0.065	0.140	0.0090
Ribosomal protein, large, P0	RPLP0	0.101	0.117	0.0118
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide	YWHAZ	0.095	0.132	0.0125
Peptidylprolyl isomerase A (cyclophilin A)	PPIA	0.127	0.110	0.0140
Low density lipoprotein receptor-related protein 10	LRP10	0.100	0.176	0.0176
Polymerase (RNA) II (DNA directed) polypeptide A, 220kDa	POLR2A	0.083	0.215	0.0179
Phosphoglycerate kinase 1	PGK1	0.163	0.136	0.0222
Actin, β	ACTB	0.101	0.239	0.0242
TATA box binding protein	TBP	0.145	0.179	0.0259
Importin 8	IPO8	0.168	0.171	0.0287
Glucuronidase, β	GUSB	0.138	0.249	0.0344
Glyceraldehyde-3-phosphate dehydrogenase	GAPDH	0.152	0.283	0.0429
Hydroxymethylbilane synthase	HMBS	0.267	0.240	0.0642
Hypoxanthine phosphoribosyltransferase 1	HPRT1	0.274	0.417	0.1144
Transferrin receptor (p90, CD71)	TFRC	0.382	0.530	0.2022
18S rRNA	18S	n.a.	0.245	n.a.
PR-BAT dataset selected putative reference genes
HECT, UBA and WWE domain containing 1	HUWE1	0.044	0.082	0.0036
Guanine nucleotide binding protein (G protein), β polypeptide 2	GNB2	0.046	0.105	0.0048
Family with sequence similarity 96, member B	FAM96B	0.063	0.090	0.0057
Proteasome (prosome, macropain) subunit, β type, 2	PSMB2	0.061	0.097	0.0059
Tumor protein, translationally-controlled 1	TPT1	0.064	0.098	0.0063
Guanine nucleotide binding protein (G protein), β polypeptide 1	GNB1	0.060	0.105	0.0063
Transmembrane protein 248	TMEM248	0.046	0.151	0.0069
Nuclear receptor binding protein 1	NRBP1	0.053	0.136	0.0072
SCY1-like 1 (S. cerevisiae)	SCYL1	0.056	0.131	0.0074
ADP-ribosylation factor 1	ARF1	0.062	0.123	0.0076
PT-BAT data set selected putative reference genes
Ring finger protein 1	RING1	0.065	0.069	0.0045
Family with sequence similarity 96, member B	FAM96B	0.063	0.090	0.0057
Proteasome (prosome, macropain) subunit, β type, 2	PSMB2	0.061	0.097	0.0059
Guanine nucleotide binding protein (G protein), β polypeptide 1	GNB1	0.060	0.105	0.0063
GNAS complex locus	GNAS	0.081	0.091	0.0074
ADP-ribosylation factor 1	ARF1	0.062	0.123	0.0076
Ring finger protein 1	RING1	0.065	0.127	0.0082
RNA binding protein S1, serine-rich domain	RNPS1	0.079	0.110	0.0087
Transcription factor 25 (basic helix-loop-helix)	TCF25	0.077	0.115	0.0089
ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R)	ELAVL1	0.073	0.122	0.0089

*Gene symbols marked in bold text were selected for qPCR analysis.

**CV-score is the product of the CV values from the PT-BAT and PR-BAT datasets.

n.a., not available.

Figure 1. Evaluation of stability- and M-values for reference genes in adipose tissue (AT) using qPCR. The evaluation was performed in 2 sets of adipose tissue samples. The first set (A and B) included 20 samples of perirenal AT with half of them containing variable levels of brown adipocytes. The second set (C and D) included the previously analyzed preirenal AT samples as well as 20 s.c. AT samples from both lean and obese subjects. qPCR results from both sets of samples were analyzed using 2 different algorithms; the normfinder algorithm (A and C) and the geNorm algorithm (B and D). Gene symbols in bold text are classified as commonly used reference genes.

Table 2. Characteristics of the study participants

Samples	BMI (Mean± SD)	Age (Mean± SD)	Gender (Males/Females)	Biopsy procedure
Perirenal (UCP1+)	25.7 ± 2.2	42.4 ± 13.8	6M/4F	Surgery
Perirenal (UCP1−)	25.8 ± 3.3	44.0 ± 9.2	6M/4F	Surgery
Subcutaneous (Lean)	22.5 ± 1.0	35.2 ± 6.8	5M/5F	Needle
Subcutaneous (Obese)	35.1 ± 3.3	40.2 ± 5.5	5M/5F	Needle

Table 3. List of candidate reference genes with gene symbol, Assay ID and mean Cq value

Gene Symbol	Assay ID	Cq Mean (all groups)
B2M	Hs00984230_m1	21.7
UBC	Hs01871556_s1	27.0
RPLP0	Hs99999902_m1	22.9
YWHAZ	Hs03044281_g1	23.0
PPIA	Hs04194521_s1	28.5
LRP10	Hs00204094_m1	25.9
GAPDH	Hs02758991_g1	23.4
18S	Hs99999901_s1	13.3
HUWE1	Hs00948075_m1	26.2
GNB2	Hs00929275_g1	25.6
TPT1	Hs02621289_g1	21.2
RING1	Hs00968517_m1	27.2
FAM96B	Hs00211406_m1	27.5
PSMB2	Hs01002946_m1	26.2
GNB1	Hs00929799_m1	25.5