Project description:Background: Urine is a potential source of biomarkers for diseases of the kidneys and urinary tract. RNA, including microRNA, is present in the urine enclosed in detached cells or in extracellular vesicles (EVs) or bound and protected by extracellular proteins. Detection of cell- and disease-specific microRNA in urine may aid early diagnosis of organ-specific pathology. In this study, we applied barcoded deep sequencing to profile microRNAs in urine of healthy volunteers, and characterized the effects of sex, urine fraction (cells vs. EVs) and repeated voids by the same individuals. Results: Compared to urine-cell-derived small RNA libraries, urine-EV-derived libraries were relatively enriched with miRNA, and accordingly had lesser content of other small RNA such as rRNA, tRNA and sn/snoRNA. Unsupervised clustering of specimens in relation to miRNA expression levels showed prominent bundling by specimen type (urine cells or EVs) and by sex, as well as a tendency of repeated (first and second void) samples to neighbor closely. Likewise, miRNA profile correlations between void repeats, as well as fraction counterparts (cells and EVs from the same specimen) were distinctly higher than correlations between miRNA profiles overall. Differential miRNA expression by sex was similar in cells and EVs. Conclusions: miRNA profiling of both urine EVs and sediment cells can convey biologically important differences between individuals. However, to be useful as urine biomarkers, careful consideration is needed for biofluid fractionation and sex-specific analysis, while the time of voiding appears to be less important.
Project description:MicroRNA (miRNA) biomarkers for fragile X syndrome were searched by urine microRNA (miRNA) profiling using deep sequencing. The urine miRNA profile of twin boys who shared the same environment but one had a FXS full mutation and the other carried a premutation allele was compared based on the similar sequence reads. The urine of twin boys showed 28 differentiatially regulated miRNAs when 219 reliable identified miRNAs were compared.
Project description:The method DFI-seq was developed to enable identification of differentially expressed genes in uropathogenic E. coli strain UTI89 during growth in human urine and in bladder epithelial cells. By utilising this new method, the aim was to identify novel virulence genes in UTI89. DFI-seq is a combination of differential fluorescence induction (DFI) with next-generation sequencing. DFI-seq was compared to DFI by analysing gene expression of UPEC in human urine and thereby confirming that DFI-seq gives a better overview of gene expression. DFI-seq was hereafter used to look at gene expression in UTI89 while infecting bladder epithelial cells. We demonstrate the usefulness of DFI-seq for identification of genes required for optimal growth of UPEC in human urine, as well as potential virulence genes upregulated during infection of bladder epithelial cells. DFI-seq holds potential for the study of bacterial gene expression in live-animal infection systems.
Project description:Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra. Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases. We performed scRNA-seq on voided urine samples. 50–100 mL middle stream urine samples were collected from 12 Chinese healthy adults and combined for droplet-based single-cell RNA sequencing after flow cytometric sorting of live cells. We presented the first single-cell atlas of adult human urine and identified multiple previously unrecognized cell types. Based on our scRNA-seq analysis data, a SOX9+ cell population was identified in adult human urine which we speculated to have progenitor potential.
Project description:The aim of the present study was to identify novel DNA methylation markers in bladder cancer (BCa) through genome-wide profiling of bladder cancer cell lines and subsequent MSP screening in urine samples. Experimental Design: MBD methylCap/seq was carried out to screen differentially methylated CpG islands using two BCa cell lines (5637 and T24) and two normal bladder mucosa (BM) samples. The top one hundred most hypermethylated targets were screened using Methylation Specific PCR (MSP) in small and big cohort of urine samples from BCa patients and normal controls. The diagnostic performance of the gene panel was further evaluated in different clinical scenarios. Results: In total, 1,627 gene promoter regions hypermethylated in BCa cell line were identified in genomic level methylation profiling. The followed screening procedure in clinical urine sample generated eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A) capable of differentiating BCa from normal control. Subsequent validation in a large sample size enabled the optimisation of 5 methylation targets (VAX1, KCNV1, TAL1, PPOX1 and CFTR) for BCa diagnosis with sensitivity and specificity of 86.32% and 87.13%, respectively. In addition, VAX1 and LMX1A methylation could predict the tumour recurrence. Conclusions: Tumor specific biomarkers of BCa could be established by first performing genome level methylation profiling with cell lines and then screening the potential targets in urine samples. The panel of methylated genes identified was promising for the early non-invasive detection and surveillance of BCa. MBD methylCap/seq was carried out to screen differentially methylated CpG islands using two BCa cell lines (5637 and T24), and two normal bladder tissue mix as control.
Project description:Exsome microRNA stably present in various body fluids (such as amniotic fluid, breast milk, blood, bronchial lavage, malignant ascites fluid, tears, saliva, and urine) shown to be associated with various pathological conditions. We report the microRNA expression profiles in porcine serum, plasma, semen, urine and bile exsome at postnatal 180-days-old by a deep sequencing technology.
Project description:To elucidate the function of 30Kc19α-Lin28A protein in osteogenic differentiation of urine-derived stem cells, we established urine-derived stem cell lines differentiated with or without protein treatment. We then performed gene expression profiling analysis using data obtained from RNA-seq of osteogenic differentiated urine-derived stem cells with or without 30Kc19α-Lin28A protein treatment, undifferentiated urine-derived stem cells, and human osteoblasts
Project description:Mid-stream urine was collected from bladder cancer patients prior to surgery. Both tumor tissue and normal bladder mucosa that are located at >3cm away from the tumor edge were obtained by cystoscopy. For the normal controls with haematuria, urine samples were collected from patients who had normal cystoscopic finding and absence of malignancy with >6 months follow-up. All urine samples were centrifuged at 2500 r.c.f. for 20 minutes and the urine supernatant was collected. Total RNA of urine supernatant and frozen tissue was extracted using MirVanaTM PARISTM Kit (Ambion) in accordance with the manufacturerâs recommended protocols. AgilentTM Human miRNA Microarray Chip (Release 13.0, Agilent Technologies, Santa Clara, CA, USA) was used to determine the microRNA expression profiles of the samples.