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:To identify the miRNA expressing profiles of Platelet microparticles(PMPs, we have employed the Agilent Human miRNA 8×60K (Design ID:046064) microarray. Platelet microparticles. The platelets were derived from citrated blood of healthy human donors under an Institutional Review Board-approved protocol. Platelets were isolated after centrifugation of blood (1200r for 30 min at 21℃), then the supernatant (platelet-rich plasma) was centrifuged at 2000r for 30 min at 21℃, and the pellet containing platelets was resuspended in RPMI-1640 medium (HyClone, Logan, UT). Platelets were counted (Clinical Laboratory, Shanghai First Maternity and Infant Hospital, Shanghai) and adjusted to a density of 150 × 106 cells/mL before supplement with 1.5% ACD(sigma ) and stimulated with thrombin (1.0 u/mL; Takeda Austria) for 1 h. PMPs were in the supernatant after centrifugation at 4000r for 10 min at 4℃,then the supernatants were centrifuged at 50,000 × g for 60 min at 4 °C. The pellets containing MPs were resuspended in RPMI-1640 medium and quantified by BCA method.
Project description:The aim of this project was to elucidate the microRNA profile of platelets and of platelet-derived microparticles isolated from clinical platelet concentrates treated or not with pathogen reduction technologies.
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: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:To understand the the effect of microparticles conjugated with vascular endothelial growth factor (VEGF) on human endothelial cells derived from human umbilical cord blood (UCB) CD34+ hematopoietic stem cells, we have employed microRNA microarray profiling. The microparticles used in this study were purchased from Invitrogen (Dynal magnetic microparticles coated with streptavidin, 4.5 micron in size) and modified in our lab. Cell suspensions were mixed with blank microparticles (without VEGF), soluble VEGF or VEGF-conjugated microparticles and seeded as hanging drops to prepare endothelial cell aggregates. Cell aggregates without any treatments were used as control. After 2 h or 8 h, the cell aggregates were collected and miRNA expression profiles were detected.