Serial Analysis Of Gene Expression Libraries Research Articles

Serial analysis of gene expression in adenocarcinoma samples and normal colonic mucosa in a Chinese population.

The goal of this study was to identify the differences in gene expression between adenocarcinoma samples and colonic normal mucosa of a Chinese population. Gene expression libraries of adenocarcinoma and normal colonic mucosa were generated by serial analysis of gene expression (SAGE). Expression tags were obtained from sequencing results using the SAGE2000 software. Representative genes of tags were determined based on the SAGEmap from NCBI. Expressed genes were selected and their expression in different libraries was compared using the SPSS 17.0 software. The expression of 78 tags representing 88 types of genes showed significant differences. Compared with normal mucosa, 38 genes showed decreased expression in cancer, whereas the expression of the remaining 50 genes showed significant up-regulation. Expressed tags in SAGE libraries of normal and cancerous tissues were significantly different. Further studies examining these genes, which showed different expression levels between the 2 tissues, may be used as tumor markers and offer clues for studying the etiopathogenesis of colon cancer.

Feature selection based on closed frequent itemset mining: A case study on SAGE data classification

Cancer is curable if it can be detected early. One way to detect cancer is by analyzing the change in expression of genes in the suspected tissue. Serial analysis of gene expression (SAGE) is a sequencing technique used for measuring the expression levels of genes. Cancer detection problem can be posed as binary classification problem like whether a tissue is cancerous or normal. SAGE libraries contain expression levels of thousands of genes which are the features. It is impossible to consider all these features for classification and also the general feature selection algorithms are not efficient with this data. In this paper, closed frequent itemset mining is proposed as a feature selection technique for identifying a small set of features which can distinguish the two classes efficiently. The performance of the proposed technique is evaluated on SAGE data related to breast tissue and a group of 26 genes are selected as best features. Two well known classifiers, extreme learning machine (ELM) and support vector machine (SVM), are used to evaluate the effectiveness of the selected features in classification and found that the proposed method works well with these classifiers.

A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.

BackgroundLarge scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts.ResultsWe generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry.ConclusionsOur results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-725) contains supplementary material, which is available to authorized users.

Reactomes of Porcine Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) has devastated pig industries worldwide for many years. It is caused by a small RNA virus (PRRSV), which targets almost exclusively pig monocytes or macrophages. In the present study, five SAGE (serial analysis of gene expression) libraries derived from 0 hour mock-infected and 6, 12, 16 and 24 hours PRRSV-infected porcine alveolar macrophages (PAMs) produced a total 643,255 sequenced tags with 91,807 unique tags. Differentially expressed (DE) tags were then detected using the Bayesian framework followed by gene/mRNA assignment, arbitrary selection and manual annotation, which determined 699 DE genes for reactome analysis. The DAVID, KEGG and REACTOME databases assigned 573 of the DE genes into six biological systems, 60 functional categories and 504 pathways. The six systems are: cellular processes, genetic information processing, environmental information processing, metabolism, organismal systems and human diseases as defined by KEGG with modification. Self-organizing map (SOM) analysis further grouped these 699 DE genes into ten clusters, reflecting their expression trends along these five time points. Based on the number one functional category in each system, cell growth and death, transcription processes, signal transductions, energy metabolism, immune system and infectious diseases formed the major reactomes of PAMs responding to PRRSV infection. Our investigation also focused on dominant pathways that had at least 20 DE genes identified, multi-pathway genes that were involved in 10 or more pathways and exclusively-expressed genes that were included in one system. Overall, our present study reported a large set of DE genes, compiled a comprehensive coverage of pathways, and revealed system-based reactomes of PAMs infected with PRRSV. We believe that our reactome data provides new insight into molecular mechanisms involved in host genetic complexity of antiviral activities against PRRSV and lays a strong foundation for vaccine development to control PRRS incidence in pigs.

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas

Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. Computer-generated genomic analysis tools identified 13,722 and 14,993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.

Effects of acupuncture on the gene expression profile of lung tissue from normal rats

Acupuncture has been demonstrated to be an effective treatment for various diseases. However, little attention has been paid to its physiological influences, especially on the changes in protein and mRNA levels following acupuncture treatment under normal conditions. In this study, we investigated the gene expression profile of lung tissue from acupuncture-treated normal rats and attempted to characterize the underlying mechanisms of the changes in expression. Three common acupoints, Dazhui (GV14), fengmen (BL12) and feishu (BL13) were selected for analysis, and 2 serial analyses of gene expression (SAGE) tag libraries of the lung tissues that were derived from the normal and acupuncture-treated rats were established. Bioinformatic analyses were carried out using the functional annotation tools of the database for annotation, visualization and integrated discovery (DAVID), the gene ontology (GO) Tree Machine and the Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. In total, 144 tags were differentially expressed (P<0.05), and the DAVID functional classification of genes demonstrated that the genes were divided into 6 types. Furthermore, GO Tree Machine analysis of the gene categories indicated that 10 enriched GO categories had become enriched after acupuncture, and that 15 KEGG pathways matched the differentially expressed tags of the 2 SAGE libraries. Our results show that the essential effects of acupuncture on normal rats include the regulation of macromolecular biosynthesis, transportation and metabolism. Cellular biosynthesis and cellular lipid metabolism are the common biological processes that occur in response to acupuncture under normal and morbid conditions, which may be the general physiological effects of acupuncture.

Abstract LB-91: Transcriptome analysis of esophageal squamous cell carcinoma by SAGE identifies a new biomarker, NRD1

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. Because most ESCC is diagnosed at advanced stages, the overall 5-year survival rate of ESCC still remains low in spite of the use of various treatment modalities. Therefore, identification of new diagnostic markers and therapeutic targets for ESCC is important. Serial Analysis of Gene Expression (SAGE) is one of the common comprehensive methods to analyze transcript expression levels, and enables the quantitative and simultaneous analysis of a large number of transcripts. To identify potential diagnostic markers and therapeutic targets for ESCC, the SAGE library was generated from one ESCC sample. A total of 14,430 tags were generated, including 5,765 that were unique. By comparing SAGE tags from the ESCC sample with those from normal human squamous esophagus, several genes that is upregulated in ESCC compared with normal squamous esophagus in ESCC were identified. Among these, we focused on the nardilysin (N-arginine dibasic convertase, NRD1) gene. NRD1 has been reported to be associated with ectodomein shedding of TNF-α through activation of ADAM proteases. There is only one report in the literature mentioning the association of NRD1 with cancer (breast cancer), however, there is no report that links expression of NRD1 and ESCC. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a high level of NRD1 expression in three of five esophageal cancer cell lines and seven of 14 ESCC samples, but not in 14 normal systemic tissues. Western blot analysis also showed upregulation of NRD1 protein in total five esophageal cancer cell lines. Immunohistochemical analysis revealed expression of NRD1 in cytoplasm of 43 out of 109 ESCC cases (39%), as well as heart, skeletal muscle, and testis in agreement with previous reports. In clinicopathological analysis, expression of NRD1 correlated with the advanced T grade, N grade and tumor stage, and was an independent predictor of survival in patients with ESCC. Knockdown of NRD1 in TE5 cells inhibited cell invasion ability. Furthermore, qRT-PCR analysis showed a reduction of matrix metalloproteinase-2 (MMP-2) expression in NRD1 knocked down cells. In conclusion, NRD1 is an independent predictor of survival, and contribute to cell invasion through regulation of MMP-2 in ESCC. NRD1 could be a novel diagnostic and therapeutic target for ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-91. doi:1538-7445.AM2012-LB-91

Methodology optimizing SAGE library tag-to-gene mapping: application to Leishmania

BackgroundLeishmaniasis are widespread parasitic-diseases with an urgent need for more active and less toxic drugs and for effective vaccines. Understanding the biology of the parasite especially in the context of host parasite interaction is a crucial step towards such improvements in therapy and control. Several experimental approaches including SAGE (Serial analysis of gene expression) have been developed in order to investigate the parasite transcriptome organisation and plasticity. Usual SAGE tag-to-gene mapping techniques are inadequate because almost all tags are normally located in the 3'-UTR outside the CDS, whereas most information available for Leishmania transcripts is restricted to the CDS predictions. The aim of this work is to optimize a SAGE libraries tag-to-gene mapping technique and to show how this development improves the understanding of Leishmania transcriptome.FindingsThe in silico method implemented herein was based on mapping the tags to Leishmania genome using BLAST then mapping the tags to their gene using a data-driven probability distribution. This optimized tag-to-gene mappings improved the knowledge of Leishmania genome structure and transcription. It allowed analyzing the expression of a maximal number of Leishmania genes, the delimitation of the 3' UTR of 478 genes and the identification of biological processes that are differentially modulated during the promastigote to amastigote differentiation.ConclusionThe developed method optimizes the assignment of SAGE tags in trypanosomatidae genomes as well as in any genome having polycistronic transcription and small intergenic regions.

Serial Analysis of Gene Expression in the Chicken Otocyst

The inner ear arises from multipotent placodal precursors that are gradually committed to the otic fate and further differentiate into all inner ear cell types, with the exception of a few immigrating neural crest-derived cells. The otocyst plays a pivotal role during inner ear development: otic progenitor cells sub-compartmentalize into non-sensory and prosensory domains, giving rise to individual vestibular and auditory organs and their associated ganglia. The genes and pathways underlying this progressive subdivision and differentiation process are not entirely known. The goal of this study was to identify a comprehensive set of genes expressed in the chicken otocyst using the serial analysis of gene expression (SAGE) method. Our analysis revealed several hundred transcriptional regulators, potential signaling proteins, and receptors. We identified a substantial collection of genes that were previously known in the context of inner ear development, but we also found many new candidate genes, such as SOX4, SOX5, SOX7, SOX8, SOX11, and SOX18, which previously were not known to be expressed in the developing inner ear. Despite its limitation of not being all-inclusive, the generated otocyst SAGE library is a practical bioinformatics tool to study otocyst gene expression and to identify candidate genes for developmental studies.

Analysis of allelic differential expression in the human genome using allele-specific serial analysis of gene expression tags

Recent reports have demonstrated that a significant proportion of human genes display allelic differential expression (ADE). ADE is associated with phenotypic variability and may contribute to complex genetic diseases. Here, we present a computational analysis of ADE using allele-specific serial analysis of gene expression (SAGE) tags representing 1295 human genes. We identified 472 genes for which unequal representation (>3-fold) of allele-specific SAGE tags was observed in at least one SAGE library, suggesting the occurrence of ADE. For 235 out of these 472 genes, the difference in the expression level between both allele-specific SAGE tags was statistically significant (p < 0.05). Eleven candidate genes were then subjected to experimental validation and ADE was confirmed for 8 out of these 11 genes. Our results suggest that at least 25% of the human genes display ADE and that allele-specific SAGE tags can be efficiently used for the identification of such genes.

HSAGEing: An Improved SAGE-Based Software for Identification of Human Tissue-Specific or Common Tumor Markers and Suppressors

BackgroundSAGE (serial analysis of gene expression) is a powerful method of analyzing gene expression for the entire transcriptome. There are currently many well-developed SAGE tools. However, the cross-comparison of different tissues is seldom addressed, thus limiting the identification of common- and tissue-specific tumor markers.Methodology/Principal FindingsTo improve the SAGE mining methods, we propose a novel function for cross-tissue comparison of SAGE data by combining the mathematical set theory and logic with a unique “multi-pool method” that analyzes multiple pools of pair-wise case controls individually. When all the settings are in “inclusion”, the common SAGE tag sequences are mined. When one tissue type is in “inclusion” and the other types of tissues are not in “inclusion”, the selected tissue-specific SAGE tag sequences are generated. They are displayed in tags-per-million (TPM) and fold values, as well as visually displayed in four kinds of scales in a color gradient pattern. In the fold visualization display, the top scores of the SAGE tag sequences are provided, along with cluster plots. A user-defined matrix file is designed for cross-tissue comparison by selecting libraries from publically available databases or user-defined libraries.Conclusions/SignificanceThe hSAGEing tool provides a combination of friendly cross-tissue analysis and an interface for comparing SAGE libraries for the first time. Some up- or down-regulated genes with tissue-specific or common tumor markers and suppressors are identified computationally. The tool is useful and convenient for in silico cancer transcriptomic studies and is freely available at http://bio.kuas.edu.tw/hSAGEing

Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis

Serial analysis of gene expression (SAGE) profiles, from posterior and median cells of the silk gland of Bombyx mori, were analyzed and compared, so as to identify their respective distinguishing functions. The annotation of the SAGE libraries was performed with a B. mori reference tag collection, which was extracted from a novel set of Bombyx ESTs, sequenced from the 3′ side. Most of the tags appeared at similar relative concentration within the two libraries, and corresponded with region-specific and highly abundant silk proteins. Strikingly, in addition to tags from silk protein mRNAs, 19 abundant tags were found (≥0.1%), in the median cell library, which were absent in the posterior cell tag collection. With the exception of tags from SP1 mRNA, no PSG specific tags were found in this subset class. The analysis of some of the MSG-specific transcripts, suggested that middle silk gland cells have diversified functions, in addition to their well characterized role in silk sericins synthesis and secretion.

Expression of distinct RNAs from 3′ untranslated regions

The 3′ untranslated regions (3′UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3′UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3′UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3′UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3′UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3′UTRs, as well as caution in the use of 3′UTR sequence probes to analyze gene expression.

Tissue-specific transcript annotation and expression profiling with complementary next-generation sequencing technologies

Next-generation sequencing is excellently suited to evaluate the abundance of mRNAs to study gene expression. Here we compare two alternative technologies, cap analysis of gene expression (CAGE) and serial analysis of gene expression (SAGE), for the same RNA samples. Along with quantifying gene expression levels, CAGE can be used to identify tissue-specific transcription start sites, while SAGE monitors 3′-end usage. We used both methods to get more insight into the transcriptional control of myogenesis, studying differential gene expression in differentiated and proliferating C2C12 myoblast cells with statistical evaluation of reproducibility and differential gene expression. Both CAGE and SAGE provided highly reproducible data (Pearson's correlations >0.92 among biological triplicates). With both methods we found around 10 000 genes expressed at levels 2 transcripts per million (0.3 copies per cell), with an overlap of 86%. We identified 4304 and 3846 genes differentially expressed between proliferating and differentiated C2C12 cells by CAGE and SAGE, respectively, with an overlap of 2144. We identified 196 novel regulatory regions with preferential use in proliferating or differentiated cells. Next-generation sequencing of CAGE and SAGE libraries provides consistent expression levels and can enrich current genome annotations with tissue-specific promoters and alternative 3′-UTR usage.

Blood cells RNA biomarkers as a first long‐term detection strategy for EPO abuse in horseracing

Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping control. In this context, gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been conducted on seven thoroughbreds treated with Eprex. This functional genomic method has been performed from total blood cells collected from each animal to assess the mRNA expression consecutive to rHuEPO injections. Sample pooling was chosen as a powerful, cost-effective, and rapid means of identifying the most common and specific changes in terms of gene expression profile and to eliminate individual variation. Consequently, three SAGE libraries were constructed, before, during, and after Eprex treatment. More than 71 440 mRNA signatures were observed and subjected to statistical analysis; 49 differentially expressed genes were identified and analyzed by qPCR. From the selected gene list, were defined as potential biomarkers in terms of their low inter-individual variation and capacity as strong markers of rHuEPO administration up to 60 days after the beginning of the doping period. In this paper, a new strategy is proposed to the horseracing industry to prevent rHuEPO abuse.

Initial Characterization ofC16orf89, A Novel Thyroid-Specific Gene

Thyroid hormone is prerequisite for proper fetal and postnatal neurodevelopment, growth, and metabolism. Although much progress has been made in the characterization of genes implicated in thyroid development and function, the majority of genes involved in this process are still unknown. We have previously applied serial analysis of gene expression (SAGE) to identify novel genes preferentially expressed in the thyroid, and this has resulted in the characterization of DUOX2 and IYD (also known as DEHAL1), two genes encoding essential enzymes in the production of thyroid hormone. In the current study we characterize the gene C16orf89, which is linked to another thyroid-specific SAGE tag CCAGCTGCCT. We establish tissue-specific expression of C16orf89 using novel tissue-specific SAGE libraries and quantitative polymerase chain reaction. In addition, we characterize the C16orf89 gene and protein, and analyze its mRNA expression in response to thyrotropin and during mouse development. C16orf89 is predominantly expressed in human thyroid tissue with a specificity intermediate between thyroid transcription factors and proteins involved in thyroid hormone synthesis. C16orf89 shows the same expression pattern as Nkx2-1 (thyroid transcription factor 1) from embryonic day (E) 17.5 onward in the developing mouse thyroid and lung. The developmental timing of C16orf89 mRNA expression is similar to that of the iodide transporter Slc5a5 (also known as Nis). Both transcripts are detected from E17.5 in the developing thyroid. This is clearly later than the onset of Tg mRNA expression (from E14.5), while Nkx2-1 and Iyd mRNA can already be detected in the E12.5 thyroid. In in vitro cell culture C16orf89 expression is stimulated by thyrotropin. The major splice variant encodes a 361 amino acid protein that is well conserved between mammals, contains an N-terminal signal peptide, is secreted in a glycosylated form, and does not contain any known functional domain. We present a novel gene highly expressed in thyroid that encodes a currently enigmatic protein.

Abstract 2178: Serial analysis of gene expression (SAGE) of esophageal squamous cell carcinoma: ADAMTS-16 is up-regulated in esophageal squamous carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. To identify potential targets for diagnosis and therapeutics of ESCC, we performed Serial Analysis of Gene Expression (SAGE) on the ESCC sample. We generated the SAGE library from one ESCC sample and obtained a total of 14,430 tags, including 5,765 that were unique. By comparing SAGE tags from ESCC samples with those from normal human squamous esophagus (Gene Expression Omnibus accession number, GSM52501), we found several genes and tags that were differentially expressed between ESCC and normal squamous esophagus. Among these, we focused on the ADAM metallopeptidase with thrombospondin type 1 motif, 16 (ADAMTS16) gene. ADAMTS has been described as part of a family of zinc dependent proteases (metzincin family), which play important roles in a variety of normal and pathological conditions including arthritis and cancer. Although ADAMTS16 is expressed in some organs, the relationship with cancers, including ESCC, has not been studied. Quantitative reverse transcription-polymerase chain reaction analysis showed a high level of ADAMTS16 expression in 8 out of 20 ESCC samples (40%) but not in 15 kinds of normal tissues. We also investigated the mRNA expression levels of SCCA1, which encodes SCC antigen. High levels of SCCA1 mRNA expression were found in 4 of 20 cases (20%). We then investigated the relation of ADAMTS16 expression to clinicopathologic characters and found that ADAMTS16 overexpression correlated with the advanced T grade (p = 0.0215). Western blot analysis revealed that ADAMTS16 protein expression was detected in all 4 ESCC cases, whereas in 1 of the 4 corresponding non-neoplastic mucosa. Furthermore, ADAMTS16 protein was detected in culture media from the TE5 ESCC cell line which expressed significant level of ADAMTS16 mRNA, indicated that ADAMTS16 is surely secreted. To evaluate the biological significance of ADAMTS16 in ESCC, we performed MTT assay and transwell invasion assay. These examination revealed that knockdown of ADAMTS16 by siRNA in TE5 cells inhibited both cell growth and invasion ability. These results suggest that ADAMTS16 has a high potential as a serum tumor marker and therapeutic target for ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2178.

Deep SAGE analysis of the Caenorhabditis elegans transcriptome

We employed the Tag-seq technique to generate global transcription profiles for different strains and life stages of the nematode C. elegans. Tag-seq generates cDNA tags as does Serial Analysis of Gene Expression (SAGE), but the method yields a much larger number of tags, generating much larger data sets than SAGE. We examined differences in the performance of SAGE and Tag-seq by comparing gene expression data for 13 pairs of libraries. We identified genes for which expression was consistently changed in long-lived worms. Additional genes emerged in the deeper Tag-seq profiles, including several ‘signature’ genes found among those zup-regulated in long-lived dauer larvae (cki-1, aak-2 and daf-16). Fifty to sixty percent of the genes differentially expressed in daf-2(−) versus daf-2(+) adults had fragmentary or no functional annotation, suggesting the involvement of as yet unstudied pathways in aging. We were able to distinguish between changes in gene expression associated with altered genotype or altered growth conditions. We found 62 cases of possible mRNA isoform switching in the 13 Tag-seq libraries, whereas the 13 SAGE libraries allowed detection of only 15 such occurrences. We observed strong expression of anti-sense transcripts for several mitochondrial genes, but nuclear anti-sense transcripts were neither abundant nor consistently expressed among the libraries.

Validation of the libraries of the serial analysis of gene expression by the application of real-time quantitative polymerase chain reaction

To validate and supplement the libraries of serial analysis of gene expression (SAGE) by the application of the real-time quantitative polymerase chain reaction PCR). The primers were designed based on the full sequences of the genes. Nine single matched tags, 6 multiple matched tags, 1 non-matched tag due to the update of the National Center for Biotechnology Information (NCBI) database, and 2 non-matched tags were selected to fulfill the validation of real-time PCR. The genes were all specifically amplified by the primers pairs. The expressions of the single matched tags were identical to those of the SAGE libraries; however, the expressions of only 3 genes of the 6 multi-matched tags were identical to those of the SAGE libraries. The PCR data of the non-matched tag due to the update of the NCBI database were opposite to those of the SAGE libraries. The data did not support the significant difference of the non-matched gene of the SAGE libraries. Real-time PCR is a reliable tool for the validation of high through-put data such as SAGE. The reliability of data depends on the match of the tags of the SAGE libraries.

Comparison of gene expression profiling between bovine subcutaneous and intramuscular adipose tissues by serial analysis of gene expression

The deposition of intramuscular adipose fat is an important factor in the quality of beef, particularly in Korean cattle and Japanese cattle. A better understanding of the differential mechanisms regulating the deposition and release of fat between intramuscular and external adipose tissues, particularly subcutaneous adipose tissues, would prove vital in understanding the deposition of intramuscular fat. A SAGE (serial analysis of gene expression) method was used to identify functionally active genes in adipose tissues and to compare the global gene expression profile between the subcutaneous and the intramuscular adipose tissues in Korean cattle. We detected 11268 tags, representing 3761 transcripts in the SAGE library of the subcutaneous adipose tissues. We detected 10830 tags, representing 2918 transcripts in the intramuscular adipose tissues. Comparison of gene expression patterns between subcutaneous adipose tissues and intramuscular adipose tissues revealed that 82 genes were differentially expressed at >2-fold levels. Among them, expression levels of 50 genes were higher in subcutaneous adipose tissues compared with intramuscular tissues, whereas those of 32 genes were higher in intramuscular adipose tissues. The genes that displayed >2-fold higher expression in subcutaneous adipose tissues included secreted protein acidic and rich in cysteine, annexin II, thyroid hormone receptor interactor 1, ferritin heavy chain polypeptide 1, cofilin, diacylglycerol O-acyltransferase and thymosin beta 4. Those that showed >2-fold higher expression levels in intramuscular adipose tissues included troponin t3, heat shock 70 kDa protein 1 and NMT2 (N-myristoyltransferase 2). Expression patterns of NMT2 and troponin t3 genes were examined during differentiation of 3T3-LI preadipocytes. NMT2 gene was actively expressed at both the growing stage of preadipocytes and the initial stage of differentiation of 3T3-L1 adipocytes, but its expression was reduced at the active stage of adipocyte differentiation. Troponin t3 gene was not expressed at the growing stage of preadipocyte, but it was actively expressed during differentiation of adipocytes. This implies that troponin t3 may play a positive role in intramuscular fat deposition of beef. Our SAGE experiment provides a differential gene expression profile in subcutaneous and intramuscular adipose tissues of Korean cattle. This information will be useful in developing method(s) to produce high-marble beef.