Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome

Dinel, Stéphanie; Bolduc, Carl; Belleau, Pascal; Boivin, A.; Yoshioka, Mayumi; Calvo, Ézéquiel; Piedboeuf, Bruno; Snyder, Eric E.; Labrie, Fernand; St‐Amand, Jonny

doi:10.1093/nar/gni025

Cited by 56 publications

(59 citation statements)

References 27 publications

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“…Here, SAGE was used to obtain a prospective overview of cellular processes that are rapidly The proportion of tags matching with sequences in GenBank and UniGene databases reported here is consistent with other SAGE studies (11,19,20). Moreover, the reproducibility of SAGE methodology has been established previously by us (21) and by others (22). Furthermore, consistency of the differentially expressed tags identification in the four independent libraries corroborates the validity and reproducibility of SAGE results.…”

Section: Discussionsupporting

confidence: 86%

Identification of Cellular Processes That Are Rapidly Modulated in Response to Tracheal Occlusion Within Mice Lungs

et al. 2008

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Lung development progresses through a process reliant on mechanical cell stretch. However, this process is not well defined at the molecular level. Our goal was to globally analyze the transcriptome of fetal mouse lungs following short periods of tracheal occlusion (TO) to identify cellular processes that are rapidly modulated in response to intraluminal stretch increase. Serial analysis of gene expression (SAGE) was used to examine the global transcriptomic response of mouse prealveolar stage lungs to in vivo TO at 1 and 3 h. SAGE results were extended by histo-and immunochemical examination. Based on the 97 TO-modulated transcripts identified, our results further point out that continuous stretch in developing lungs leads directly to rapid and highly specific phenotypic modifications in a significant proportion of pulmonary cells. We conclude that intraluminal stretch increase during prealveolar stage of lung development induces a critical transition of pulmonary cells phenotype in which there is an increase in ␣-smooth muscle actin A t the end of gestation, fetal lung volume is under the influence of an important and continuous distention that is secondary to the continuous intraluminal liquid secretion by the fetal lung epithelium (1). Within developing lungs, this liquid sequestration occurs by the active larynx closure during the apnea (1), which accounts for the major portion of late fetal life (2). Abnormal loss of lung liquid is detrimental to lung development (3). Indeed, fetal cervical cord transection, which results in a lack of coordination between larynx opening and diaphragmatic movements, thus allowing excessive loss of lung liquid during fetal breathing movements, leads to lung hypoplasia (4). Similarly, loss of larynx tonus (5) and bypass of the larynx by experimental tracheotomy (6) during fetal life are both associated with excessive lung liquid loss and severe lung hypoplasia. In contrast, stimulation of lung growth and development can be achieved by using tracheal obstruction techniques at an appropriate developmental stage (7). By sequestering intraluminal liquid, fetal tracheal occlusion (TO) amplifies continuous stretch. TO represents a unique in vivo methodology that facilitates examination of the natural three-dimensional mechanical forces effect on lung development.Although the relationship between the degree of fetal lung expansion and lung development is well established, the mechanisms involved are not well understood. We hypothesize that an increase in fetal lung stretch causes a stimulus that rapidly mediates gene expression before any change in fetal lung structure is observed. To investigate the mechanisms underlying stretch-induced lung maturation, we previously created an in vivo murine model of TO that demonstrated a significant structural acceleration of prealveolar stage lung growth and development in a 24-h period (8). With this model, we recently demonstrated that TO induces an immediate cellular response (9). Indeed, when compared with sham-TO and unoperated-o...

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Section: Discussionsupporting

confidence: 86%

Identification of Cellular Processes That Are Rapidly Modulated in Response to Tracheal Occlusion Within Mice Lungs

et al. 2008

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“…Sequence files were analyzed using the SAGEana program, a modification of SAGEparser (St-Amand et al, Quebec, PQ, Canada;Dinel et al 2005). In brief, SAGE tags corresponding to linker sequences were discarded and replicate concatemers were counted only once.…”

Section: Bioinformatic Analysismentioning

confidence: 99%

“…We have previously shown that the SAGE method is very reproducible with r 2 Z0 . 96 between two SAGE libraries generated from two cDNA libraries constructed from the same total RNA pool (Dinel et al 2005). Classification of the transcripts was based upon the updated information of the genome directory (Adams et al 1995) found at the TIGR web site (http://www.tigr.…”

Section: Bioinformatic Analysismentioning

confidence: 99%

Gender difference of androgen actions on skeletal muscle transcriptome

Yoshioka¹,

Boivin²,

Bolduc³

et al. 2007

Journal of Molecular Endocrinology

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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.

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“…Although other techniques such as DNA microarray are limited by their ability to analyze only previously known transcripts, SAGE does not require a priori knowledge of the sequence of mRNA transcripts expressed in the tissues of interest. In addition, we recently showed (22) that the SAGE method has a very high reproducibility, with r 2 ϭ 0.96. Moreover, no mRNA species had significant difference in their level of expression estimated by two SAGE libraries constructed from the same pool of total RNA (22).…”

mentioning

confidence: 99%

Regulation of hypothalamic gene expression by glucocorticoid: implications for energy homeostasis

Nishida

Yoshioka

St‐Amand

2006

Physiological Genomics

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Using the serial analysis of gene expression (SAGE) method, we studied the effects of adrenalectomy (ADX) and GC on the transcriptomes of mouse hypothalamus. Approximately 180,000 SAGE tags, which correspond to 50,000 tag species, were isolated from each group of intact or adrenalectomized mice as well as 1, 3, and 24 h after GC injection. ADX upregulated diazepam binding inhibitor gene expression while downregulating vomeronasal 1 receptor D4, genes involved in mitochondrial phosphorylation (cytochrome-c oxidase 1 and NADH dehydrogenase 3), 3␤-hydroxysteroid dehydrogenase-1, and prostaglandin D2 synthase. GC increased the gene expression levels of dehydrogenase/reductase member 3, prostaglandin D2 synthase, solute carrier family 4 member 4, and five cytoskeletal proteins including myosin light chain phosphorylatable fast and troponin C2 fast. On the other hand, GC reduced the mRNA levels of calmodulin 1 and expressed sequence tag similar to calmodulin 2, ATP synthase F0 subunit 6, and solute carrier family 4 member 3. Moreover, 7 uncharacterized and 43 novel transcripts were modulated by ADX and GC. The present study has identified genes that may regulate hypothalamic systems governing energy balance in response to ADX and GC.transcriptome; serial analysis of gene expression; adrenalectomy; obesity CLINICAL FEATURES OF ANOREXIA and weight loss in patients with adrenal insufficiency as well as increased food intake and weight gain in patients with glucocorticoid (GC) excess implicate adrenal GC in energy homeostasis (18). Obesity is dependent on GC action, because rodent models of obesity with pathologies of genetic (13), dietary (65), and hypothalamic (38) origins are normalized by adrenalectomy (ADX) and restored by GC replacement (27). In normal rodents and humans, GC increases food intake and body weight, as well as inducing metabolic features of obesity, such as insulin resistance, hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia (30, 76). Interestingly, an administration of leptin, which barely affects body weight and food intake, becomes powerful and long-lasting in ADX rats, suggesting that GC antagonizes the central action of leptin (75). GC not only increases food intake but also decreases energy expenditure by suppressing thermogenesis in mouse brown adipose tissue (63). Thus GC promotes positive energy balance, whereas a lack of GC is linked to hypophagia and reduced body weight.The hypothalamus is a brain center that regulates energy homeostasis by integrating peripheral signals such as leptin, insulin, and GC. Thus the effects of ADX and GC on hypothalamic gene expression have been a major focus of many previous studies (45,54,60,73). However, no study has previously investigated effects of ADX and GC in the transcriptome of the hypothalamus. The serial analysis gene expression (SAGE) method accurately measures the expression levels of tens of thousands of genes, previously known or not, and finds the genes related to diseases or the effects of stimuli (59,68). Although other techniques ...

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Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome

Cited by 56 publications

References 27 publications

Identification of Cellular Processes That Are Rapidly Modulated in Response to Tracheal Occlusion Within Mice Lungs

Identification of Cellular Processes That Are Rapidly Modulated in Response to Tracheal Occlusion Within Mice Lungs

Gender difference of androgen actions on skeletal muscle transcriptome

Regulation of hypothalamic gene expression by glucocorticoid: implications for energy homeostasis

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