Project description:This project analyzes pancreatic tissue profiles of pancreatic cancer patients, pancreatitis patients, and controls. Since miRNAs are known to be valuable diagnostic markers, we asked whether respective patterns of pancreatic cancer patients can be detected in biopsies. The project aimed at an impoved understanding of complex profiles rather than single markers. Thus, a high-throughput technique was necessary, profiling all known miRNAs integratively. Three markers have been validated by using qPCR. n = 22 normal controls, n = 27 pancreatitis samples, and n = 136 pancreatic cancer samples have been screened for the complete miRNA repertoire. Please note that each miRNA has been measured in seven replicates and the median of the replica has been computed.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
Project description:Pancreatic cancer has a dismal prognosis partly due to late diagnosis, where a reliable non-invasive diagnosis is urgently wanted. We here explored, whether serum-derived PaCa exosomes may provide a diagnostic means and microRNA may be advantageous. Exosomes were collected from the serum of 133 patients with pancreatic cancer, 22 patients with non-malignant pancreatic tumors, 25 patients with chronic pancreatitis and 20 healthy donors. Exosomes were isolated by ultracentrifugation and used qRT-PCR for miRNA quantification and miRCURY LNA microRNA Array for miRNA analysis.
Project description:This project analyzes pancreatic tissue profiles of pancreatic cancer patients, pancreatitis patients, and controls. Since miRNAs are known to be valuable diagnostic markers, we asked whether respective patterns of pancreatic cancer patients can be detected in biopsies. The project aimed at an impoved understanding of complex profiles rather than single markers. Thus, a high-throughput technique was necessary, profiling all known miRNAs integratively. Three markers have been validated by using qPCR.