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 samples[1].In order to confirm GDI as house-keeping 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.
Figure 1. Raw data expression of LRP10, GDI and CIDEA
Figure 2. Expression of CIDEA normalized by LRP10 and GDI
[1]GabrielssonBG, 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.