Project description:26 breast cancer patient serum EV derived RNA and 4 control serum EV derived RNA was sequenced to explore the diagnostic potential of EV. EV RNA was isolated by EXOQUICK® Exosome isolation and RNA purification kit for serum/plasma (System Biosciences,CA) and was reverse transcribed to make amplified cDNA by SMART seq v4 ultra-low input RNA kit (Takara Bio Inc, USA). A sequencing library was made using 1 ng of sheared cDNA using The Low Input Library Prep Kit v2 (Takara Bio Inc, USA). DNA unique Dual index kit was used to combine libraries for sequencing (Takara Bio Inc, USA). NOVASEQ 6000 was used for sequencing to generate 25 million paired end reads per sample.
Project description:The overall goal of the study was to determine if serum or serum EV microRNAs added prognostic value to current clinical nomograms for prostate cancer. Serum was collected from 203 patients with biopsy-proven prostate cancer. EVs were isolated from the 132 serum samples. RNA was isolated from these serum and serum EV samples, and 61 microRNAs were quantified per sample on a custom qPCR plate. The microRNA data was normalized using NormFinder. The normalized microRNA data was then compared to adverse pathology and prostate biopsy Gleason grade group.
Project description:Purpose: The goals of this study are to compare the serum extracellular vesicle (EV) delivered miRNA levels of patients with bone-metastatic prostate cancer (PCa), non-bone -metastatic PCa and benign prostatic hyperplasia (BPH), and to identify EV-delivered microRNAs in patient’s serum as indicators for bone-metastatic PCa. Methods:Serum extracellular vesicle delivered miRNA profiles of patients with bone-metastatic PCa or non-bone -metastatic PCa or BPH were generated by deep sequencing, using Illumina HiSeqTM 2500 platform Results: Using an optimized data analysis method, we mapped about 17 million sequence reads per sample. Differential analysis showed the expressions of 35 EV delivered miRNAs were significantly different between serum of patients with PCa and BPH, with a p value <0.05. the expressions of 5 EV delivered miRNAs were confirmed with qRT–PCR. Conclusions: Serum EV-delivered miR-181a-5p is a promising diagnostic biomarker for bone-metastatic PCa.
Project description:Extracellular Vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, we isolated EV from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV) and mature (CMm-EV) cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV were characterized in terms of expression of specific markers, yield, and size. Bioactivity was assessed in human umbilical vein endothelial cells (HUVEC) and hiPSC-CM. Small RNA-Seq was performed to identify the differentially expressed miRNA in the four EV groups. Bioactivity assays showed increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increased hiPSC-CM proliferation. Global miRNA expression profiles corroborated an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification. A stemness maintenance miRNA cluster upregulated in hiPSC-EV was found to target the PTEN/PI3K/AKT pathway. Moreover, hiPSC-EV treatment mediated PTEN suppression and increased AKT phosphorylation. Overall, our findings validate hiPSC as suitable cell biofactories for EV production for cardiac regenerative applications.
Project description:In the research field of extracellular vesicles (EVs), the use of EV-depleted fetal bovine serum (FBS) for in vitro studies is highly recommended to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or bought commercially, nevertheless these depletion methods do not guarantee an RNA-free preparation. In this study we have addressed the RNA contamination issue in FBS, ultracentrifuged EV-depleted FBS, commercially available EV-depleted FBS, and also from our recently developed filtration based EV depleted FBS. Commercially available serum-free, xeno-free defined media were also screened for RNA contamination.
Project description:With the goal to identify miRNAs and other small RNAs involved in EV-mediated oligodendrocyte-neuron communication, we performed deep sequencing (RNA-seq) of small RNAs derived from EVs isolated from culture supernatants of primary mouse oligodendrocytes by differential centrifugation. Oligodendrocytes were cultured and EVs harvested under serum-free conditions in presence of B27 supplement. We found that RNA isolated from EVs harvested from cells under serum-replacement conditions contains miRNA contaminants carried into the sample by defined media components.
Project description:Purpose: The goals of this study are to compare the serum extracellular vesicle (EV) delivered miRNA levels of patients with bone-metastatic prostate cancer (PCa), non-bone -metastatic PCa and benign prostatic hyperplasia (BPH), and to identify EV-delivered microRNAs in patient’s serum as indicators for bone-metastatic PCa. Methods:Serum extracellular vesicle delivered miRNA profiles of patients with bone-metastatic PCa or non-bone -metastatic PCa or BPH were generated by miRNA chip array, using Agilent-070156 Human_miRNA_V21.0_Microarray plateform. Results: Differential analysis showed the expressions of 27 EV delivered miRNAs were significantly different between serum of patients with bone-metastatic PCa and non-bone-metastatic PCa with a p value <0.05. the expressions of 5 EV delivered miRNAs were confirmed with qRT–PCR. Conclusions: Serum EV-delivered miR-181a-5p is a promising diagnostic biomarker for bone-metastatic PCa.