Project description:We have adapted a commercial assay that employs mRNA quantification based on the frequency of PCR amplicons determined by next-generation to a high-throughput semi-conductor sequencing platform (Ion-Torrent Proton). We show parallel amplification of pathway derived transcript sets/genes in 12 reference RNA samples followed by sequence-based quantification covering a dynamic range of five orders of magnitude with low technical variation.
Project description:We have adapted a commercial assay that employs mRNA quantification based on the frequency of PCR amplicons determined by next-generation to a high-throughput semi-conductor sequencing platform (Ion-Torrent Proton). We show parallel amplification of pathway derived transcript sets/genes in 12 reference RNA samples followed by sequence-based quantification covering a dynamic range of five orders of magnitude with low technical M-BM- variation. Expression of selected genes were profiled at four time points (0d, 10d, 20d, and 60d) during differentiation of induced pluripotent stem cells into cardiomyocytes, with three independent biological replicates at each time point.
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:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
