Supplementary information on housekeeping gene
LRP10 is another housekeeping gene introduced in our lab because it has been identified as a gene
with the least variation in expression levels in adipose samples1. In order to confirm GDI as housekeeping gene in the present experimental setting-up and to check lower expression in females
compared with males, additional assays were performed.
From the original RNA sample, we prepared a new working dilution, followed by a RT reaction and
PCRs of three genes. In Supplementary Figure 1 we present the whole set of raw data obtained (Ct
values) of the two housekeeping genes (LRP10 and GDI) and CIDEA. Both housekeeping genes show
a small range of variation between the different groups, being LRP10 more constant than GDI. In
absolute terms and without any normalisation, CIDEA shows higher Ct values in females, and an
opposite pattern to males in comparison with both GDI and LRP10. In addition, CIDEA expression
after normalization is also lower in females (S, p=0.000), irrespective of the housekeeping gene
used (Supplementary Figure 2). Therefore, these data confirm our results about CIDEA, which is
clearly expressed at lower levels in females than in males.
Differences associated to dietary treatment were also present under both housekeeping genes (D,
p=0.037 in CIDEA/LRP10 and p=0.019 in CIDEA/GDI).
Although there are specific dissimilarities between the two sets of data (these new ones and the
ones enclosed in the manuscript), which in part could be associated to the number of samples (one
was lost in this working dilution), all the above results (Supplementary Figure 1 and 2) are very
close to our original data (see Figure 2 in the manuscript) which also identified a significant sex
effect (p=0.000) and of dietary treatment (p=0.031). Altogether, we believe that this additional
assay provide confirmation that CIDEA is less expressed in females versus males and that GDI (or
LRP10) are representative housekeeping genes in these samples.
The manuscript keep the original data set because expression of GDI and of the rest of the genes
analysed have been obtained from the same working dilution and treated at the same time and
under the same experimental conditions.
1
Gabrielsson BG, Olofsson LE, Sjögren A, Jernås M, Elander A, Lönn M, Rudemo M, Carlsson LM. Obes Res. 2005
Apr;13(4):649-52. Evaluation of reference genes for studies of gene expression in human adipose tissue.
Figure 1. Raw data expression of LRP10, GDI and CIDEA
Figure 2. Expression of CIDEA normalized by LRP10 and GDI