Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Comparing two transcriptome technologies - sequencing match microarrays [RNA-Seq]

ABSTRACT: Next-generation sequencing has emerged as a promising platform for whole genome transcriptome profiling. The microarray technology has in contrast a long track record, but may suffer from issues related to background fluorescence and non-specific hybridization. The two technologies have been compared for eukaryotic transcriptomes in various studies. However, for prokaryotes information on comparative performance of the two technologies is sparse. We thus compared the two technologies for Streptococcus thermophilus. With just single experiments there was a strong correlation between the technologies. Two samples were analyzed

ORGANISM(S): Streptococcus thermophilus

SUBMITTER: Martin Pedersen

PROVIDER: E-GEOD-34477 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.

Project description:This experiment contains the RNA-Seq samples only. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5 % success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80 % of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Project description:Puberty is a complex physiological event measured by various indicator traits in genetic improvement programs of beef cattle; thus, developing a more complete understanding of the genes and regulatory pathways and networks involved in puberty will provide knowledge to help improve genetic selection strategies. Herein, we characterized the transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to growth and metabolism needed for cattle to achieve puberty (i.e., longissimus dorsi muscle, fat, and liver). These tissues were collected from pre (PRE)- and post (POST)-pubertal Brangus (3/8 Brahman; Bos indicus x 5/8 Angus; Bos taurus) heifers derived from a population of cattle used to identify QTL associated with fertility traits. In order to exploit the power of complementary omics analyses, PRE and POST puberty co-expression gene networks were constructed by combining the results from RNA-Seq, GWAS, and bovine transcription factors. RNA-Seq of 8 tissues among PRE and POST Brangus heifers revealed 1515 differentiallyexpressed and 943 tissue-specific genes within the 17,832 genes confirmed by metrics of RNA-Seq analysis. Combining the results from RNA-Seq and GWAS indentified a total of 25 QTL associated to heifer fertility. The hypothalamus experienced the most notable up-regulation of genes via puberty. Complementary omics procedures revealed 2,450 co-expressed genes across the 8 tissues relative to puberty. The PRE network had 372,861 connections whereas the POST network had 328,357 connections. A sub-network from this process revealed key transcriptional regulators (i.e., PITX2, FOXA1, TSG1D1, DACH2, LHX4, PROP1 and SIX6). Results from multiples sources of omics data will facilitate the design of breeding strategies to improve fertility in Bos indicus-influenced composite cattle. Sixty-one samples from PRE and POST pubertal composite beef heifers were analyzed with RNA-Seq. The transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to metabolism andbody morphometrics needed for cattle to achieve puberty (i.e.,) was characterized. These tissues were collected from pre (PRE)- and post (POST)-pubertal Brangus (3/8 Brahman x 5/8 Angus) heifers derived from a population of cattle used to identify QTL associated with fertility. Total RNA was purified using a Trizol protocol (Invitrogen, Carlsbad, CA). Sequencing libraries were made using TruSeq RNA Sample Preparation kit of Illumina (San Diego, CA).

Dataset Information

Comparing two transcriptome technologies - sequencing match microarrays [RNA-Seq]

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure