Project description:Transcriptome analysis of sus1 mutant in reference to its parental wild type reference. Iterative global median method was used for between sample normalization. z-test was performed to evaluate differential gene expression. Keywords: repeat sample
Project description:The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant extracellular vesicles (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics as sample EV and function as an internal quantitative and qualitative control throughout analysis. Spike-in applications of rEV in bodily fluids prior to EV analysis map technical variability of EV applications and promote intra and inter laboratory studies. This protocol describes the production, recovery and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on fluorescence-, nucleic acid- and protein measurements. Multiple application potentials for rEV are exemplified, including method development, big data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4–5 d.
Project description:Despite serving as a central experimental technique in epigenetics research, chromatin immunoprecipitation (ChIP) suffers from several serious drawbacks: it is a relative measurement untethered to any external scale that obviates fair comparison amongst experiments; it employs antibody reagents that have differing affinity and specificity for target epitopes, which are in turn variable in abundance; and it is frequently not reproducible. To address these problems, we developed internal standard calibrated ChIP (ICeChIP), a method of spiking a native chromatin sample with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons and revealing a correlation between the apparent symmetry of H3K4me3 in promoter nucleosomes and gene expression. Direct in situ assessment of immunoprecipitation accommodates for a number of experimental pitfalls, and provides a critical examination of untested assumptions inherent in conventional ChIP.
Project description:Isobaric labeling has the promise of combining high sample multiplexing with precise quantification. However, normalization issues and the missing value problem of complete n-plexes hamper quantification across more than one n-plex. Here we introduce two novel algorithms implemented in MaxQuant that substantially improve the data analysis with multiple n-plexes. First, isobaric matching between runs (IMBR) makes use of the three-dimensional MS1 features to transfer identifications from identified to unidentified MS/MS spectra between LC-MS runs in order to utilize reporter ion intensities in unidentified spectra for quantification. On typical datasets, we observe a significant gain in quantifiable n-plexesMS/MS spectra that can be used for quantification. Second, we introduce a novel PSM-level normalization, applicable to data with and without common reference channel. It is a weighted median-based method, in which the weights reflect the number of ions that were used for fragmentation. On a typical dataset, we observe complete removal of batch effects and dominance of the biological sample grouping after normalization. This dataset is one of the datasets used for the study. It is TMT 10-plex with a reference channel.
Project description:Despite serving as a central experimental technique in epigenetics research, chromatin immunoprecipitation (ChIP) suffers from several serious drawbacks: it is a relative measurement untethered to any external scale that obviates fair comparison amongst experiments; it employs antibody reagents that have differing affinity and specificity for target epitopes, which are in turn variable in abundance; and it is frequently not reproducible. To address these problems, we developed internal standard calibrated ChIP (ICeChIP), a method of spiking a native chromatin sample with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons and revealing a correlation between the apparent symmetry of H3K4me3 in promoter nucleosomes and gene expression. Direct in situ assessment of immunoprecipitation accommodates for a number of experimental pitfalls, and provides a critical examination of untested assumptions inherent in conventional ChIP. Examination of spiked-in semi-synthetic nucleosomes in ICeChIP-seq experiments performed for HEK293, mESC E14 and DM S2 cell line
Project description:Targeted mRNA expression profiling was performed using nCounter® Tumor Signaling 360™ Panel (NanoString Technologies) composed of 780 genes (including internal reference genes) involved in cellular energetics, sustained proliferation, evasion of growth suppression, genomic instability, resistance to cell death, epithelial to mesenchymal transition (EMT), and metastasis. Normalization of housekeeping genes for the quantification of gene expression levels, normalization of the positive control for background correction, and data analysis were performed using the nSolverTM analysis software (NanoString Technologies).
Project description:Epigenomic profiling by ChIP-seq is a prevailing methodology used to investigate chromatin-based regulation in biological systems, such as human disease, yet the lack of an empirical methodology to normalize amongst experiments has limited the usefulness of this technique. Here we describe a “spike-in” normalization method that allows the quantitative comparison of histone modification status across cell populations using defined quantities of a reference epigenome. We demonstrate the utility of this method in measuring epigenomic changes following chemical perturbations and show how control normalization of ChIP-seq experiments enables discovery of disease-relevant changes in histone modification occupancy.
Project description:Mice over-expressing human PITX2A in cornea by the keratocan upstream regulatory sequences. Normalization procedure: Data normalized to spikes. Values below 0.01 were set to 0.01. All of the genes in each sample were divided by the median of a user specified list of positive control genes (chip-to-chip normalization). Keywords: repeat sample
Project description:To characterize the translation defects in Maf1-/- samples, we used ribosome profiling on three WT and three Maf1-/- fed livers. Each sample was spiked with a fixed amount of Drosophila S2 Schneider cell material as an internal control for sample-to-sample normalization, and to allow detection of global changes in translation.