Project description:Since microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human eye tissue using miRNA-Seq in order to provide a foundation for future disease research. Total RNAs were extracted from normal human ciliary body (CB) (n=7), cornea (n=7), and trabecular meshwork (TM) (n=7) samples using mirVana total RNA isolation kit. The sequencing library was prepared using the Illumina TruSeq Small RNA Sample Prep kit and was sequenced using Illumina MiSeq. Generated sequence reads were trimmed and aligned against human reference database using the Bowtie software, and only exact matches to mature miRNAs from miRBase were included. miRTarBase database was used to analyze the gene targets of the miRNAs in our tissues, and expression of a few selected miRNAs were validated using droplet digital PCR (ddPCR). We found 378 miRNAs expressed collectively in our samples, of which miR-143-3p, miR-184, miR-26a-5p, and miR-204-5p were most abundantly expressed. With the expression patterns and gene targets, we identified several uniquely expressed miRNAs and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we were able to validate the expression profile created using miRNA-Seq. For the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Knowing the expression of miRNAs in non-diseased eye tissues could help elucidate miRNA changes that accompany diseases such as glaucoma and keratoconus. This study profiled the expression of miRNAs in normal human ciliary body (n=7), cornea (n=7), and trabecualr meshwork (n=7) tissue
Project description:Since microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human eye tissue using miRNA-Seq in order to provide a foundation for future disease research. Total RNAs were extracted from normal human ciliary body (CB) (n=7), cornea (n=7), and trabecular meshwork (TM) (n=7) samples using mirVana total RNA isolation kit. The sequencing library was prepared using the Illumina TruSeq Small RNA Sample Prep kit and was sequenced using Illumina MiSeq. Generated sequence reads were trimmed and aligned against human reference database using the Bowtie software, and only exact matches to mature miRNAs from miRBase were included. miRTarBase database was used to analyze the gene targets of the miRNAs in our tissues, and expression of a few selected miRNAs were validated using droplet digital PCR (ddPCR). We found 378 miRNAs expressed collectively in our samples, of which miR-143-3p, miR-184, miR-26a-5p, and miR-204-5p were most abundantly expressed. With the expression patterns and gene targets, we identified several uniquely expressed miRNAs and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we were able to validate the expression profile created using miRNA-Seq. For the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Knowing the expression of miRNAs in non-diseased eye tissues could help elucidate miRNA changes that accompany diseases such as glaucoma and keratoconus.
Project description:MicroRNA expression in the mouse eye.MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina . This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures.
Project description:Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 16,015 nuclei in human adult testis. This dataset includes five samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.
Project description:Introduction: In human eyes, ocular enlargement/ growth, reflects active scleral extracellular matrix remodeling. miRNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences, and serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses miRNAs, some of which modulate genes regulating ocular growth. In this study, the scleral miRNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses. Results: Human sclera expressed several miRNAs. Microarray comparison of adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10^11), with increased expression of collagen specific mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 in fetal sclera subsequently confirmed (1.5 to 4 fold changes, p<0.01). For both adult and fetal samples, no significant differences in miRNA expression profiles of sclera from posterior and peripheral ocular regions were observed. Conclusion: This is the first study to catalogue miRNA expression in human sclera. The sclera expresses several miRNAs, some of which show age-related differential regulation, higher in rapidly growing fetal eyes, consistent with a role in ocular growth regulation. These findings may be useful for linking scleral miRNA expression with potential manipulation in disorders such as scleral ectasia/ axial myopia.
Project description:Affymetrix GeneChip miRNA arrays were used to profile the expression of microRNAs in three ATCC renal cell-lines and a pool of normal human tissues (hREF, first choice human total RNA, Ambion).Intra-platform concordance among technical replicates of a pool of normal human tissues (hREF), three renal cell lines (A498, Caki-2 and HK-2) and three cell lines belonging to NCI-60 human cell lines panel publicly available on Array Express repository, were considered.