Project description:Ratiometric controls were prepared from commercial sources of high quality RNA from human tissues with distinctly different expression patterns. The samples were processed using six different target labeling protocols and replicate datasets were generated on high density gene expression microarrays. The area under the curve from receiver operating characteristic plots was calculated as a measure of diagnostic performance. The reliable region of the dynamic range was derived from log ratio deviation plots made for each dataset. These samples and metrics provide a mechanism for assessing process improvement that can be used by clinical laboratories conducting microarray assays. <br><br>Additional processed data files are available on the FTP site for this experiment in E-TABM-1091.additional.zip.
Project description:The link between the gut microbiota and the human physiological state has been demonstrated in recent years. High gut microbiota diversity has been linked to many beneficial functions necessary or human health, while dysbiosis has been correlated to different pathological states. In this context, the study of the gut microbiota results of high relevance been necessary the development of different techniques capable of characterizing this complex ecosystem. Metaproteomics has been proved useful in the characterization of complex protein samples becoming a suitable tool for the study of these microbial communities. However, due to the complexity of these samples, protein extraction protocols may affect metaproteomics results. In this context, we evaluated stool sample processing (SSP) and microbial cell disruption, assessing the impact of different protocol modifications in the number of peptides and proteins identified. We compared different stool processing conditions and microbial cell disruption methods in terms of protein and peptide identifications and taxonomic profiles.
Project description:As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification kits applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored.
Project description:Anti-FLAG-affinity purification of Umbrea associated factors from nuclear extracts of Drosophila Schneider cells expressing FLAG-HA-Umbrea. Anti-FLAG -affinity purifications from Schneider cell nuclear extracts not expressing any FLAG-tagged protein served as a control SDS-PAGE separting both samples, 9 slices per sample, reduction and alkylation, digestion with trypsin. LC-MS on a Ultimate3000-LTQ Orbitrap XL Raw data were analysed using the MaxQuant 1.2.2.5 software package. Identified proteins were considered as interaction partners if their MaxQuant intensities displayed a greater than 10 fold enrichment compared to control anti-FLAG purifications from L2-4 nuclear extracts not expressing any FLAG-tagged protein
Project description:Considerable variation in gene expression data from different DNA microarray platforms has been demonstrated. However, no characterization of the source of variation arising from labeling protocols has been performed. To analyze the variation associated with T7-based RNA amplification/labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using 3 eukaryotic target preparation methods and hybridized to a single array type (Affymetrix U95Av2). Variability was measured in yield and size distribution of labeled products, as well as in the gene expression results. All methods showed a shift in cRNA size distribution, when compared to un-amplified mRNA, with a significant increase in short transcripts for methods with long IVT reactions. Intra-method reproducibility showed correlation coefficients >0.99, while inter-method comparisons showed coefficients ranging from 0.94 to 0.98 and a nearly two-fold increase in coefficient of variation. Fold amplification for each method was positively correlated with the number of present genes. Two factors that introduced significant bias in gene expression data were observed: a) number of labeled nucleotides that introduces sequence dependent bias, and b) the length of the IVT reaction that introduces a transcript size dependent bias. This study provides evidence of amplification method dependent biases in gene expression data.