Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.
Project description:We have designed and experimentally validated the BactoChip, a 60-mer oligonucleotide microarray for simultaneous detection and quantification of multiple bacterial species of clinical interest. The Bactochip microarray targets a novel set of high-resolution marker genes, those genes that most unequivocally characterized each bacterial species. The accuracy of the BactoChip microarray was evaluated using the labeled total DNA of single bacterial species at different concentrations (from 65ng to more than 250ng). The specificity of the developed array was further validated using mixed cultures containing up to 15 different bacterial species in even or staggered amount. We employed the Agilent 'Custom HD-CGH 8x15k Array" (catalogue number: G4427A) and the Agilent'Genomic DNA ULS labeling Kit" (catalogue number: 5190-0419). The microarray successfully distinguished among bacterial species from 21 different genera. The BactoChip additionally proved accurate in determining species-level relative abundances over a 10-fold dynamic range in complex bacterial communities. In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. We analyzed to which extend the universal reference can be used as a tool for the relative quantification of miRNAs across multiple experiments. We compared the results of direct hybridizations i.e. sample vs. sample to those of indirect hybridizations i.e. sample vs. UR. For the direct hybridizations, we hybridized 5µg liver total RNA vs 5 µg brain total RNA (n = 3) and for the indirect hybridization 5 µg liver or brain total RNA vs UR (5 fmol/miRNA) (n = 3). We calculated the so-called re-ratios for the UR experiments by dividing the signal ratios of the liver vs. UR array by the respective brain vs. UR array gaining a liver vs. brain re-ratio. Each RNA sample was mixed with 5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled. The RNA mix was hybridized in a dual colour approach to microarrays. The mean ratios of all probes were normalized to the median of the ratios detected for the spiked 18 synthetic RNA oligonucleotides reverse complement to miRControl 3 probes.
Project description:Three 2cm segments were excised from different parts (TOP, MID, BOT) along the vertical axis of a 4 week old stem of hemp (C. sativa), and the outer layers of the stem were compared using a cDNA amplicon array. Each segment represented a different developmental stage, especially in relation to bast fibre differentiation (i.e. TOP= elongation, MID=transition, BOT= thickening). Only the cDNAs that showed the highest differential expression were sequenced.
Project description:Balancing the competing demands of phagolysosomal degradation and autophagy is a significant challenge for phagocytic tissues. Yet, how this plasticity is accomplished in health and disease is poorly understood. In the retina, circadian phagocytosis and degradation of photoreceptor outer segments by the postmitotic retinal pigment epithelium (RPE) is essential for healthy vision. Disrupted autophagy due to mTOR overactivation in the RPE is associated with blinding macular degenerations; however, outer segment degradation is unaffected in these diseases, indicating that distinct mechanisms regulate these clearance mechanisms. Here, using advanced imaging and mouse models, we identify optineurin as a key regulator that tunes phagocytosis and lysosomal capacity to meet circadian demands and helps prioritize outer segment clearance by the RPE in macular degenerations. High-resolution live-cell imaging implicates optineurin in scissioning outer segment tips prior to engulfment, analogous to microglial trogocytosis of neuronal processes. Optineurin is essential for recruiting LC3, which anchors outer segment phagosomes to microtubules and facilitates phagosome maturation and fusion with lysosomes. This dynamically activates transcription factor EB (TFEB) to induce lysosome biogenesis in an mTOR-independent, TRPML1 (transient receptor potential-mucolipin 1)-dependent manner. RNAseq analyses show that expression of TFEB target genes temporally tracks with optineurin recruitment, and that lysosomal and autophagy genes are controlled by distinct transcriptional programs in the RPE. The unconventional plasma membrane-to-nucleus signaling mediated by optineurin ensures outer segment degradation under conditions of impaired autophagy in macular degeneration models. Independent regulation of these critical clearance mechanisms would help safeguard metabolic fitness of the RPE through the organismal lifespan.
Project description:Balancing the competing demands of phagolysosomal degradation and autophagy is a significant challenge for phagocytic tissues. Yet, how this plasticity is accomplished in health and disease is poorly understood. In the retina, circadian phagocytosis and degradation of photoreceptor outer segments by the postmitotic retinal pigment epithelium (RPE) is essential for healthy vision. Disrupted autophagy due to mTOR overactivation in the RPE is associated with blinding macular degenerations; however, outer segment degradation is unaffected in these diseases, indicating that distinct mechanisms regulate these clearance mechanisms. Here, using advanced imaging and mouse models, we identify optineurin as a key regulator that tunes phagocytosis and lysosomal capacity to meet circadian demands and helps prioritize outer segment clearance by the RPE in macular degenerations. High-resolution live-cell imaging implicates optineurin in scissioning outer segment tips prior to engulfment, analogous to microglial trogocytosis of neuronal processes. Optineurin is essential for recruiting LC3, which anchors outer segment phagosomes to microtubules and facilitates phagosome maturation and fusion with lysosomes. This dynamically activates transcription factor EB (TFEB) to induce lysosome biogenesis in an mTOR-independent, TRPML1 (transient receptor potential-mucolipin 1)-dependent manner. RNAseq analyses show that expression of TFEB target genes temporally tracks with optineurin recruitment, and that lysosomal and autophagy genes are controlled by distinct transcriptional programs in the RPE. The unconventional plasma membrane-to-nucleus signaling mediated by optineurin ensures outer segment degradation under conditions of impaired autophagy in macular degeneration models. Independent regulation of these critical clearance mechanisms would help safeguard metabolic fitness of the RPE through the organismal lifespan.
Project description:The photoreceptor outer segment is the canonical example of a modified and highly specialised cilium, with an expanded membrane surface area in the form of discs or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localisation of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display a reduction in shed outer segment material in the RPE at 1 month post fertilisation (mpf), but otherwise normal retinal structure and visual function up to 12 mpf. Cone photoreceptorspecific transgenic reporter lines show Rab28 localises almost exclusively to outer segments, independently of nucleotide binding. Co-immunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, Phosphodiesterase 6C and Guanylate Cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.
Project description:Mutations in the POMT1 gene, encoding a protein O-mannosyltransferase essential for alpha-dystroglycan (α-DG) glycosylation, are frequently observed in a group of rare congenital muscular dystrophies, collectively known as dystroglycanopathies. However, it is hitherto unclear whether the effects seen in affected patients can be fully ascribed to α-DG hypoglycosylation. To study this, we here used comparative mass spectrometry-based proteomics and immunofluorescence microscopy, in order to investigate the changes in retina of mice in which Pomt1 is specifically knocked out in photoreceptor cells. Our results demonstrate significant proteomic changes and associated structural alteration in photoreceptor cells of Pomt1 cKO mice. In addition to effects related to impaired α-DG O-mannosylation, we observed morphological impairments in the outer segment that are associated with dysregulation of a relatively understudied POMT1 substrate (KIAA1549), BBSome proteins and retinal stress markers. In conclusion, our study provides new hypotheses to explain the phenotypic changes that are observed in the retina of patients with dystroglycanopathies.