A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling
ABSTRACT High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis.
This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system.
These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/. S3T source code and datasets can also be downloaded from the aforementioned website.
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ABSTRACT: HTLV-1 is the etiologic agent of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD8+ T cells may contribute to the protection or development of HAM/TSP. In this study we used SAGE methodology to screen for differentially expressed genes in CD8+ T cells isolated from HTLV-1 asymptomatic carriers (HAC) and from HAM/TSP patients, in order to identify genes involved in HAM/TSP development. SAGE analysis was conducted by pooling samples according to clinical status. The comparison of gene expression profiles between HAC and HAM/TSP libraries identified 285 differentially expressed tags. We focus on cytotoxicity and cytokines related genes due to their potential biological role in HTLV-1 infection. Our results showed that patients with HAM/TSP have high expression levels of degranulation related genes, namely GZMH and PRF1, and of the cytoskeletal adaptor PXN. We found that GZMB and ZAP70 were overexpressed in HTLV-infected patients compared to non-infected group. We also detected that CCL5 was higher in HAM/TSP group, as compared to HAC and to CT groups. Our findings showed that CD8+ T cells of HAM/TSP patients have an inflammatory and active profile. PXN and ZAP70 overexpression in HTLV-1 infected patients was described for the first time here and reinforce this concept. However, although active and abundant, CD8+ T cells are not able to completely eliminate infected cells and prevent the development of HAM/TSP and, moreover, these cells might contribute to the pathogenesis of the disease by migrating to CNS. These results should be further tested with biological functional assays to increase our understanding on the role of these molecules in the development of HTLV-1 related diseases.AIDS research and human retroviruses 01/2013; 29(5). DOI:10.1089/AID.2012.0205 · 2.46 Impact Factor
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ABSTRACT: ALTHOUGH THE MOLECULAR PATHOGENESIS OF PITUITARY ADENOMAS HAS BEEN ASSESSED BY SEVERAL DIFFERENT TECHNIQUES, IT STILL REMAINS PARTIALLY UNCLEAR. RIBOSOMAL PROTEINS (RP) HAVE BEEN RECENTLY RELATED TO HUMAN TUMORIGENESIS, BUT THEY HAVE NOT YET BEEN EVALUATED IN PITUITARY TUMORIGENESIS. OBJECTIVE THE AIM OF THIS STUDY WAS TO INTRODUCE SAGE (SERIAL ANALYSIS OF GENE EXPRESSION), A HIGH-THROUGHPUT METHOD, IN PITUITARY RESEARCH IN ORDER TO COMPARE DIFFERENTIAL GENE EXPRESSION. METHODS 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 TWENTY-TWO GH-SECRETING PITUITARY TUMORS AND IN FIFTEEN NORMAL PITUITARIES. RESULTS COMPUTER GENERATED GENOMIC ANALYSIS TOOLS IDENTIFIED 13,722 AND 14,993 EXCLUSIVE GENES IN NORMAL AND ADENOMA LIBRARIES, RESPECTIVELY. BOTH SHARED 6,497 GENES: 2,188 were underexpressed and 4,309 overexpressed in tumoral library. In adenoma library, 33 genes encoding ribosomal proteins were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by Real-Time PCR. Conclusion 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 down-regulated genes encoding ribosomal proteins. Altogether, the present data suggests 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.European Journal of Endocrinology 09/2012; DOI:10.1530/EJE-12-0760 · 3.69 Impact Factor