High Resolution Transcription Profiling using Next-Generation-Sequencing identifies Diagnostic Markers for Malignancy

Ralf Horres1,3, Björn Rotter1, Peter Winter1, Michalski Christoph2, Friess Helmut2, Jüngling Ruth1,3, Günter Kahl3, Nicolas Krezdorn1, Klaus Hoffmeier1, Jörg Kleeff2
1GenXPro GmbH, Germany; 2Chirurgische Klinik und Poliklinik, Klinikum rechts der Isar, TU München; 3Goethe Universität Frankfurt, Institut für Molekulare Biowissenschaften

Since up to 95% of all transcripts are expressed at low levels, these rare transcripts are extremely important for a cell‘s fate and function. A SuperTag Digital Gene Expression (ST-DGE) Profiling approach was established and applied for the analysis of malignant potential of pancreatic cancers. In contrast to microarrays ST-DGE is more reproducible, detects higher dynamic ranges of gene expression, discovers natural antisense transcripts and also counts rare transcripts, which are obscured in a microarray’s background signal. The highly reliable gene expression signatures allow choosing a subset of diagnostic transcripts converting the assay to a more clinically friendly PCR-based assay. This study aimed at analyzing processes underlying the tendency towards malignancy from chronic pancreatitis (CP) to benign intraductal papillary mucinous tumor (IPMT) to highly malignant ductal adenocarcinoma of the pancreas (PDAC). ST-DGE generated a complete survey of poly-adenylated transcripts in excised normal, CP, IPMTs and PDAC pancreatic tissues. Both the sense (s) and antisense (as) transcriptomes of CP and IPMT were significantly different from normal pancreas, and all three deviated from the transcript patterns characteristic for the tendency to malignancy, finally manifested in PDACs. A subset of these transcripts is now tested as prognostic and diagnostic qRT-PCR-based markers for malignancies in pancreatic tissue. GenXPro, est. 2005, is a service provider for the analysis of nucleotide-based information content of any biological sample. The service portfolio includes the best available, focused and sensitive techniques, mostly using next generation sequencing, delivering qualitative and quantitatively most accurate results, at lowest possible costs. GenXPro offers full-service, “from tissue to data” including most suited bioinformatics approaches for the individual next generation data handling. Our data can be conveniently analyzed using both our own “analysis suite” and /or excel. Our analyzes include quality assessment, quantification, transcriptome mapping, Genome mapping, Gene Ontology analysis, plots , etc. to ease the analysis and publications. Our technical service portfolio is headed by our patented „SuperSAGE“ and „SuperTag Digital Gene Expression“ (ST-DGE) Profiling, for ultra deep transcriptomics studies, but also includes RNAseq, normalization & sequencing of cDNA, microRNA analysis, epigenetics, copy number variation detection and genotyping as well as qPCR service. GenXPro is involved in the development of several diagnostic genetic markers for human diseases and in plant and animal breeding.

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