GNPS - fecal and serum sample extraction method test
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Pieter C. Dorrestein
PROVIDER: MSV000091948 | GNPS | Mon May 15 12:18:00 BST 2023
REPOSITORIES: GNPS
Similar Datasets
Project description:Background: During the analysis of ripening related gene expression in tomato fruit, we observed a bias towards certain classes of messenger RNA based upon the type of buffer used in the initial step of the extraction procedure. We postulated that there was a functional association of the separated transcripts. To test this hypothesis, we carried out extractions from the same tissue sample where only the buffer varied. Transcripts were hybridised to Affymetrix oligo arrays and the data was analysed according to the predicted cellular component of the encoded proteins. Results: The use of an extraction buffer that lacked high levels of caeotropic agents resulted in a reduction of mRNAs encoding proteins that were either secreted or were predicted to be routed through the endomembrane system and hence could have been bound to the endoplasmic reticulum in polyribosomes. Extraction of the cell debris immediately following the initial buffer based extraction and subsequent micro array analysis revealed that the expected transcripts could be recovered. This demonstrated that the buffer was separating the mRNAs based on the cellular component of the encoded proteins. Analysis of selected transcripts by northern hybridisation again supported this theory. Conclusions: Some traditional buffers used for fruit RNA extraction selectively deplete for transcripts encoding proteins that are membrane-associated or secreted. This can be explained if polyribosomes that are bound to the endoplasmic reticulum (ER) are not effectively disrupted when the extraction buffer lacks either detergent or organic solvents. These findings have important implications with respect to experimental bias, as well as opportunities for message enrichment and protein characterisation.
2011-01-21 | GSE17222 | GEO
Project description:Background: During the analysis of ripening related gene expression in tomato fruit, we observed a bias towards certain classes of messenger RNA based upon the type of buffer used in the initial step of the extraction procedure. We postulated that there was a functional association of the separated transcripts. To test this hypothesis, we carried out extractions from the same tissue sample where only the buffer varied. Transcripts were hybridised to Affymetrix oligo arrays and the data was analysed according to the predicted cellular component of the encoded proteins. Results: The use of an extraction buffer that lacked high levels of caeotropic agents resulted in a reduction of mRNAs encoding proteins that were either secreted or were predicted to be routed through the endomembrane system and hence could have been bound to the endoplasmic reticulum in polyribosomes. Extraction of the cell debris immediately following the initial buffer based extraction and subsequent micro array analysis revealed that the expected transcripts could be recovered. This demonstrated that the buffer was separating the mRNAs based on the cellular component of the encoded proteins. Analysis of selected transcripts by northern hybridisation again supported this theory. Conclusions: Some traditional buffers used for fruit RNA extraction selectively deplete for transcripts encoding proteins that are membrane-associated or secreted. This can be explained if polyribosomes that are bound to the endoplasmic reticulum (ER) are not effectively disrupted when the extraction buffer lacks either detergent or organic solvents. These findings have important implications with respect to experimental bias, as well as opportunities for message enrichment and protein characterisation. GeneChip analyses were performed to analyse the effect of using different extraction protocols on Solanum lycopersicum pericarp.
2011-01-21 | E-GEOD-17222 | biostudies-arrayexpress
Project description:Purpose: Description of a spike-adjusting-method (SAM) to normalize ChIP-seq data . Methods: We performed ChIP-seq of POLR3D and POLR2B with mouse liver supplemented with 2.5% of human DNA. Human DNA will be used as an internal control for ChIP-seq quantification. Results: We show that using the SAM for ChIP-seq quantification improve similarity of POLR3D and POLR2B ChIP-seq replicates samples and improve difference between samples originate from different conditions. Conclusions: The SAM improves comparison of ChIP-seq samples, either by increasing similarity between replicates or by emphasise differences between conditions. Chromatin Immuno-precipitations were performed with antibodies directed against POLR3D (Pol III) and POLR2B (Pol II) using mouse liver material supplemented with human DNA. Immuno-precipitated DNA was next sequenced using Illumina HiSeq. Three different concentrations of human spiked DNA were tested for the Pol III ChIP (2.5%, 5% and 10%). We also sequenced the corresponding inputs (crosslinked DNA from mouse liver). Two concentrations of human spiked DNA (5% and 10%) were tested for the Pol 2 ChIP. We also sequenced the corresponding inputs (crosslinked DNA from mouse liver).
2014-04-02 | E-GEOD-52049 | biostudies-arrayexpress
Project description:Compared to using dispersive SPE (dSPE) based on the QuEChERS procedure, we found similar reproducibility using high purity MgSO4 to analyze standard reference material (SRM) of human serum and human plasma samples and slightly higher recovery of targeted chemicals using MgSO4. To avoid contamination by environmental chemicals in solvents and reagents used for QuEChERS, we chose to use high purity MgSO4 to remove water-soluble interferences.
2021-05-04 | ST001787 | MetabolomicsWorkbench
Project description:DNA extraction method from alfalfa Metagenome
| PRJNA901244 | ENA
Project description:Soil is an inherently complex matrix and as such, we believe when performing culture-independent microbial community analyses using the 'omics' suite of tools, all biomolecules investigated should be co-extracted from the same biological sample. To this end, we developed a robust, cost-effective DNA, RNA and protein co-extraction method for soil. The samples deposited here represent 3 biological replicates from one of eight soil types tested in this work.
2019-07-15 | PXD007077 | Pride
Project description:These data include the genome wide occupancy of H2AUbq and H3K27me3 by ChIP sequencing in Ring1a-/-;Ring1bf/f Cdkn2a-/- MLL-AF9 leukemic cells treated with OHT or EtOH
2016-10-21 | GSE85327 | GEO
Project description:Induced phase separation extraction (IPSE) is an efficient sample clean-up technique that can replace liquid-liquid extraction (LLE). The purpose of this study was to miniaturize IPSE by carrying it out in a microfluidic chip. An IPSE chip was designed and evaluated for its ability to separate and purify samples on a microscale. The 5 × 2 cm chip was fed with a solution of polar to non-polar model compounds in acetonitrile-water (1:1). In the 100 µm wide and 40 µm deep microchannels, the sample solution was efficiently separated into two immiscible phases by adding a hydrophobic solvent as inducer. Analytes present in the sample solution each migrated to their own favorable phase upon phase separation. After optimization, extraction and fractionation were easily and efficiently achieved. The behavior of analytes with a pH-dependent partitioning could be influenced by adjusting the pH of the sample solution. Scutellaria baicalensis extract, used in Traditional Chinese Medicine (TCM), was successfully separated in aglycones and glycosides. In this microscale system, the sample and solvent consumption is reduced to microliters, while the time needed for the sample pretreatment is less than one minute. Additionally, the extraction efficiency can reach up to 98.8%, and emulsion formation is avoided.
| S-EPMC7796191 | biostudies-literature
Project description:The aim of this study is to determine if, using antioxidant drugs, it is possible to interfere with the proliferative capabilities of the human glioblastoma (GBM) tumor-initiating cells (TICs). To establish which cellular processes are activated in GBM TICs by the antioxidants NAC, Tiron and Trolox, we generated and analyzed the gene expression profiles after treatment with these compounds and with H2O and EtOH (vehicles).
2014-03-04 | E-GEOD-44099 | biostudies-arrayexpress
Project description:Background and Aims: Liver proteomics with limited sample amounts is technically challenging but valuable for innovative hepatology research. We aimed to establish a simple and high-efficient approach for protein extraction and preparation from the formaldehyde-fixed paraffin-embedded (FFPE) liver samples for microproteomics assessment. Methods: Individual cell collected by laser capture microdissection (LCM) from FFPE liver slices were used as test samples with microscale samples of fresh-frozen liver or cultured liver cells as controls. Protocol for extracting protein was determined through the orthogonal test of 6 conditions of buffer compositions and heating processes. For sample preparation, we adjusted the protocol of single-tube solid-phase sample preparation (SP3) by enhancing protein precipitation and elution and developed a new method named HDMSP. HDMSP was then tested for its capability, reproducibility, and sample-type compatibility in analysis with nanoscale liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Results: For FFPE protein extraction, adding 4% SDS significantly increased the production by 2.5 times; incubation for 2 hours at 95℃ in alkaline amine buffer improved both the efficiency and quality of protein extraction. For the development of HDMSP, using 2% SDS to elute protein, using 70% acetonitrile but not ethanol, and increasing input of carboxyl magnetic beads to 2 mg/ml improved the rate of protein recovery by 88%, 50%, and 74%, respectively. For either 20 nL FFPE liver or HepG2 cell samples, or 1~2 μg fresh-frozen liver samples, the rate of protein recovery was stable at around 75%. LC-MS/MS demonstrated that the protocol designed for FFPE samples protein extraction allowed for unbiased extraction of insoluble or hydrophobic proteins and with HDMSP, the depth, identification, physicochemical properties, subcellular locations, and reproducibility of FFPE liver microproteome was comparable to those of fresh-frozen sample control. HDMSP also showed high efficiency and reproducibility for subcellular nuclear and cytoplasm-isolated proteins. Conclusion: The new approach including 300mM Tris, 4% SDS incubation for 2h at 95°C, and then preparation with HDMSP is simple, robust, and highly efficient for use in microproteomics of FFPE liver samples. This protocol can provide a solution reference for liver FFPE microproteomics.
2023-04-25 | PXD040010 |