MicroRNA profiling of human renal tubular epithelial cells - miR-17 and miR-19b to investigate their down-regulatation in human aging
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ABSTRACT: Renal Tubular Epithelial Cells (RPTEC) from two donors were cultivated in vitro until irreversible growth arrest was observed. Total RNA from young (replicating) as well as growth arrested cells was harvested and hybridized to LNA based microRNA microarrays using a looped design. After data processing, differential expression of miRNAs in senescent vs. young RPTEC cells was calculated.
Project description:From two donors of human umbilical vein endothelial cells, in vitro cell lines were established. Both cell lines were grown in vitro until irreversible growth arrest was observed (replicative senescence). Total RNA from young (replicating) cells as well as senescent cells was harvested and used for hybridization of microRNA chips (MRC) from TU Graz based on Sanger miRBase 9.2
Project description:Renal Proximal Tubular Cells of young / senescent replicative age or high / low reactive oxygen species content were profiled using an Affymetrix Chip Platform.<br>Sample classifications: <br>Young refers to the in vitro age of early passage cells. <br>Senescent indicates cells at the end of their in vitro life span, having entered an irreversible growth arrest. <br>SortHI refers to cells with high intracellular reactive oxygen species content that were sorted by FACS according to a bright FL1 fluorescence after staining with 1 ï¾µM CM-H2DCFDA (Molecular Probes Cat No C6827).<br>SortLO refers to cells with low intracellular reactive oxygen species content that were sorted by FACS according to a dim FL1 fluorescence after staining with 1 ï¾µM CM-H2DCFDA.<br>
Project description:Normal human fibroblasts were isolated from skin biopsies of two different healthy causcasian patients, a 41-years old female (HDF-1) and a 16-years old male (HDF-5). HDF-5 and HDF-1 cells were cultivated in DMEM/HAMs F-12 medium (Biochrom KG, Berlin, Germany) supplemented with 10% fetal calf serum (FCS) 4 mM L-glutamine (Sigma) and Primocin antibiotics (100 µg/mL) grown at 37 °C in ambient atmosphere containing 5% CO2. Cells were grown until confluence was reached and routinely passaged using 1:4 or 1:3 split ratios. Upon decrease in growth rate and entry of irreversible growth arrest split ratio was changed to 1:2 until cells stopped dividing and fresh medium was added at least every 7 days. HDF-1 cells reached senescence after 54 population doublings, while HDF-5 cells had stopped growing after 72.7 population doublings (see Fig. S2 for growth curves and staining of senescent associated beta-galactosidase).<br>Senescent HDF RNA samples were compared to young HDF RNA samples in terms of microRNA expression using Exiqon LNA microRNA microarrays.
Project description:Foreskin samples were derived from young and old donors. Total RNA was extracted and used for hybridization to microRNA microarrays. From 4 young and 4 old donors, differential expression of microRNAs was analyzed using R/Bioconductor and linear models
Project description:Mesenchymal stem cells were isolated from several donors of different age (young vs old), and total RNA was extracted. Samples were labeled with Cy3 and Cy5 dyes and hybridized in a looped design that allowed the calculation of differentially expressed miRNAs in MSCs isolated from healthy, aged individuals.
Project description:T cells from several blood donors were separated in populations corresponding to CD8+ CD28+ and CD8+ CD28- surface markers. Total RNA extracts from cells were hybridized to MRC LNA microarray. Contrasts of interest were CD8CD28- minus CD8CD28+ (replicative aging) and CD8CD28+ (old donors) minus CD8CD28+ (young donors), which corresponds to chronological aging.
Project description:HBEC-5i (human brain endothelial cells) grown to confluence in 6-well tissue culture plates. Cocultures were either left unstimulated or incubated overnight with recombinant TNF?. HBEC were then washed in PBS and incubated with PLT (platelets), pRBC (P.falciparum-parasited red blood cells) or NRBC (normal red blood cells) according to the experimental conditions. Then HBEC were washed three times with PBS, and were harvested at 0 and 5h coculture. Each experimental condition was realized in triplicate.
Project description:H69 cells (human cholangiocytes) were exposed to TNF-alpha (10 ng/ml) for 8 h and were then collected for miRNA analysis, compared with non-treated control cells.