Project description:The limiting-dilution study evaluated the effects of sample dilution on the ability to identify and quantify analytes in plasma. The study was divided into 10 batches with identical experimental design spanning over a 16-day period. Each batch consisted of 33 aliquots with 11 different plasma extract volumes (0 – 700 µL) corresponding to 11 plasma concentrations repeated three times.
Project description:The objective of this work was to determine the effectiveness of cross-hybridization of gDNA from five native soil nematodes to an Affymetrix Caenorhabditis elegans tiling array. Cross-hybridization experiments using C. briggsae, for which genome information is available, allowed hybridisation intensities to be correlated with known sequence differences. Initial analysis of data by conventional array-based Comparative Genomic Hybridization (aCGH) techniques at the chip level lead to misleading results due to an artefact from the combination of scaling, bandwidth smoothing, and differential GC content in exon and intron regions. To circumvent this artefact, individual probes were instead normalized and centered by adjusting for probe-specific thermodynamic binding affinity. However, cross-hybridization of C. briggsae DNA revealed that the resultant probe intensities alone were still uncorrelated to sequence similarity below 90% identity. Below 90% similarity, all probes hybridize uniformly poorly, and above 90% similarity the hybridization differences are not large enough to detect over background, therefore, no 'threshold' ratio of hybridization intensity was successful at identifying probes with similarity to the heterologous genome. In light of the observations described here, we suggest that the criteria for replication and verification of gene expression profiles generated from cross-species microarray hybridizations be more stringent than typically adopted for con-specific hybridizations.
Project description:ngs2012_02_microd-ngs microd-part2-Effect of dilution on RNA seq sensitivity-One sample of total RNA extracted from whole embryo by laser assisted microdissection was diluted from 5ng to 10pg to check sensitivity of the library construction and subsequent sensitivity of transcript identification by RNA seq.
Project description:The objective of this work was to determine the effectiveness of cross-hybridization of gDNA from five native soil nematodes to an Affymetrix Caenorhabditis elegans tiling array. Cross-hybridization experiments using C. briggsae, for which genome information is available, allowed hybridisation intensities to be correlated with known sequence differences. Initial analysis of data by conventional array-based Comparative Genomic Hybridization (aCGH) techniques at the chip level lead to misleading results due to an artefact from the combination of scaling, bandwidth smoothing, and differential GC content in exon and intron regions. To circumvent this artefact, individual probes were instead normalized and centered by adjusting for probe-specific thermodynamic binding affinity. However, cross-hybridization of C. briggsae DNA revealed that the resultant probe intensities alone were still uncorrelated to sequence similarity below 90% identity. Below 90% similarity, all probes hybridize uniformly poorly, and above 90% similarity the hybridization differences are not large enough to detect over background, therefore, no 'threshold' ratio of hybridization intensity was successful at identifying probes with similarity to the heterologous genome. In light of the observations described here, we suggest that the criteria for replication and verification of gene expression profiles generated from cross-species microarray hybridizations be more stringent than typically adopted for con-specific hybridizations. Genomic DNA from Caenorhabditis elegans N2 (Bristol), C. elegans CB4856 (Hawaiian), C. briggsae AF16, Oscheius tipulae KS585, Oscheius FVV-2 KS555, Mesorhabditis sp. KS587, Acrobeloides sp. KS586, and Chiloplacus sp. KS584 were hybridized onto C. elegans Affymetrix tiling array (two replicate chips were performed for each species).
Project description:N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification, present on tRNA, rRNA and recently investigated in eukaryotic mRNA. We report ac4C-seq, a chemical genomic method for single-nucleotide resolution, transcriptome-wide quantitative mapping of ac4C. While we did not find detectable ac4C sites in human and yeast mRNAs, ac4C was induced via ectopic overexpression of eukaryotic acetyltransferase complexes, invariably at a conserved sequence motif. In contrast, cross-evolutionary profiling reveals unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, ncRNA and mRNA from hyperthermophilic archaea. Ac4C is dramatically induced in response to temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Cryo-EM visualization of WT and acetyltransferase-deficient archaeal ribosomes furnishes structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for unravelling this modification’s role in biology and disease.
Project description:We investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl (B6N). We sequenced mRNA extracted from liver tissue of 10 M. Musculus individuals. 4 liver samples were collected from 10 week old inbred strains (1 male and 1 female Berlin Fat Mouse Inbred line (BFMI), 1 male and 1 female C57Bl/6NCrl (B6N)) and 6 liver samples collected from 10 week old F1 males using a reciprocal cross design (3 paternal BFMI (patBFMI) vs 3 maternal BFMI (matBFMI)).
Project description:Wild type steady state chemostats cultures, in dilution rate of 0.3[1/hr] or 0.1[1/hr], and phosphate concentration in the feeding vessel of 0.3 or 0.1mM Pi