The serial analysis of gene expression (SAGE) method is used to study global gene expression in cells or tissues in various experimental conditions. However, its reproducibility has not yet been definitively assessed. In this study, we have evaluated the reproducibility of the SAGE method and identified the factors that affect it. The determination coefficient (R2) for the reproducibility of SAGE is 0.96. However, there are some factors that can affect the reproducibility of SAGE, such as the replication of concatemers and ditags, the number of sequenced tags and double PCR amplification of ditags. Thus, corrections for these factors must be made to ensure the reproducibility and accuracy of SAGE results. A bioinformatic analysis of SAGE data is also presented in order to eliminate these artifacts. Finally, the current study shows that increasing the number of sequenced tags improves the power of the method to detect transcripts and their regulation by experimental conditions.

In order to identify the potential peripheral signals of appetite and satiety from duodenum, we have performed a transcriptomic study in the mucosa after high-fat (HF) and low-fat (LF) meal ingestion. After fasting, one group of mice was killed and the others were fed ad libitum with HF or LF diet, and killed 30 min, 1 h, and 3 h after the beginning of the meal. The duodenum mucosa was sampled, and the serial analysis of gene expression (SAGE) method was performed. The mRNA regulations were confirmed by real-time PCR. Energy, protein, and fat intakes were higher in the HF than in the LF group. Gene expression profile revealed 118 characterized or partially characterized differentially expressed transcripts. The HF meal delayed the expressions of peptidases compared to the LF groups. Most of mRNAs related to fat absorption, including apolipoprotein A-IV (Apoa4), were decreased in HF1h group, whereas plasma triglyceride (TG) levels were comparable between HF and LF groups. Noteworthy, these downregulations were concomitant to a break in fat intake 1 h after HF meal. At the same time, the HF meal induced transcripts related to cell growth and organization, whereas transcripts involved in cell defense were repressed. Moreover, we have identified fat-responsive transcripts. This study has characterized the molecular responses of duodenum mucosa after HF or LF meal ingestion. Characterization of novel fat-specific candidates whose relations with feeding behavior have never been reported may contribute to the development of new therapeutic targets for appetite and satiety controls.

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50 000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBP , cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.

Adipose tissue transcriptome by serial analysis of gene expression. Obes Res. 2004;12:750 -757. Objective: To describe the genomic expression profile or transcriptome of adipose tissue using the serial analysis of gene expression method. Research Methods and Procedures:The serial analysis of gene expression strategy is based on isolation of short sequences (tags), which usually correspond to unique transcripts, and on their concatenation into long DNA molecules, which are then cloned and sequenced. Experiments were performed with mRNA from retroperitoneal adipose tissue of male C57BL6 mice. Results: We isolated 45,996 tags corresponding to more than 17,000 different genes. Eighty-eight genes were expressed at more than 0.1% of the total population and represented 26% of the mRNA population identified. The most expressed genes were: carbonic anhydrase 3 (1.97%), cytochrome c oxidase (COX) 1 (1.47%), COX2 (1.25%), diazepam binding inhibitor (1.04%), a novel transcript (0.87%), COX3 (0.55%), fatty acid-binding protein 4 (0.55%), and NADH dehydrogenase 4 (0.52%). Other genes known to be expressed in adipose tissue, such as uncoupling protein 2, angiotensinogen, adipsin, and insulin-like growth factor 1, were found at a lower level. Several tags corresponding to novel transcripts were also found.Discussion: To our knowledge, the present results provide for the first time a quantitative description of the transcriptome in adipose tissue.

Sarcopenia is related to metabolic syndrome in postmenopausal women. Hormone replacement therapies with androgens improve muscle functions by molecular mechanisms that are still unknown, at least partly because the skeletal muscle transcriptome has been less characterized in females. We performed the serial analysis of gene expression method in six experimental groups, intact (male and female), ovariectomy (OVX), OVXCdihydrotestosterone (DHT) injection 1, 3, or 24 h before kill in mice. The 438 transcript species differentially expressed between gender showed that females had higher expression levels of mRNA related to cytoskeleton/contractile apparatus and mitochondrial processes as well as protein, lipid, and amino acid metabolisms. In females, OVX and DHT modulated 109 and 128 transcript species respectively. OVX repressed transcripts of fast/glycolytic fiber, glycolysis, and glucose transport, whereas all these effects were reversed 3 h after the DHT injection. Moreover, DHT treatment induced transcripts which reduce intracellular Ca 2C level at early time points. These results may suggest that DHT treatment in OVX mice increases muscle contractility by affecting fiber distribution and intracellular Ca 2C concentration as well as improving glucose metabolism. On the other hand, transcripts of fast/oxidative fiber, oxidative phosphorylation, and ATP production were repressed 24 h after DHT administration. In our previous study using male mice, transcripts in oxidative phosphorylation and ATP production were induced 24 h after DHT injection (Yoshioka M, Boivin A, Ye P, Labrie F & St-Amand J 2006 Effects of dihydrotestosterone on skeletal muscle transcriptome in mice measured by serial analysis of gene expression. Journal of Molecular Endocrinology 36 247-259). These results demonstrate gender differences in DHT actions on skeletal muscle, and contribute to a precise understanding of the molecular mechanisms of androgen actions in the female skeletal muscle.