Real time PCR from expression microdissection (xMD) of epithelial cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE205216 | GEO | 2022/06/01
REPOSITORIES: GEO
Similar Datasets
Project description:GeoMx Digital Spatial Profiling (Nanostring) is a commercial spatial transcriptomics method to selectively analyze regions of interest within intact tissue sections. However, it is unclear how tissue decalcification affects the results of this assay, or if decalicified samples are comparable to untreated samples. For this purpose, we assembled a set of tissues that were subjected to progressive durations of incubation with ethylene-diamine-tetra-acetic (EDTA), and analyzed comparable regions of the tissue using GeoMx DSP. In the associated manuscript, we show that EDTA incubation attenuates signals, and distorts counts from a subset of probes. We determine the undisclosed full-length target sequences of probes used in the human whole transcriptome panel, updating target transcripts and genes, and identify that gene-promiscuous probes are particularly affected by normalization after EDTA incubation. For the purpose of this study, we prepared series of non-calcified tissues that were incubated with EDTA for increasing time periods, and matched control samples from the same source tissue, which were left untreated. Specimens submitted for routine tissue analysis were collected for research purposes after approval of the ethics commission of the Medical University of Vienna (2167/2021, registered at https://ekmeduniwien.at/core/catalog/2022/) as graphically shown in Figure 1A). In the course of routine pathology workup, 24 clinical tissue specimens (Table S1) of different organs stemming from 20 patients were fixed in 7.5% buffered formalin for 2.0 to 3.5 (mean 3.3, median 3.0) days according to the laboratory’s routine procedure. At macroscopic dissection, an excess piece of tissue was removed and divided into 4 macroscopically similar samples. Tissues were then either processed immediately in a Tissue-Tek VIP® 6 AI (Sakura Finetek) or incubated at room temperature in EDTA solution (Tritiplex, Glatt-Koller, 403212270) for 1, 3, and 7 days before processing them analogous to the untreated tissue. Samples were embedded in paraffin blocks according to standard protocols using low-melting point Paraffin (Histo-Comp, ATS-200856). Blocks were sectioned, stained with hematoxylin/eosin and scanned (Pannoramic 250 Flash II, 3DHistech) to confirm the content of equivalent histological structures among samples sourced from the same specimen. Areas for tissue microarray (TMA) cores were selected using the digital slide scans. Tissue microarrays were then assembled using an automated TMA device (TMA Grandmaster, 3DHistech) using 1mm punches and a recipient block of 5mm depth. TMA were tempered by alternating storage at 37°C, 4°C, and room temperature, for 5 times (2h each), following by a single incubation at 65°C for 10 minutes. Slides were sectioned using a rotating microtome at 5µm thickness. One section was used for DSP analysis as outlined below, while an adjacent section was used for validation of the TMA cores using hematoxylin & eosin stain. DSP was performed according to protocols provided by Nanostring using the following standard parameters. In brief, sections were deparaffinized and subjected to heat-induced protein epitope retrieval (using retrieval buffer at pH 9, Thermo Fisher Scientific, 00-4956-58) for 15 minutes at 100°C using a steam cooker. Retrieval for mRNA was performed using proteinase K (Thermo Fisher Scientific, 25530049) at 1µg/ml for 15 minutes. Slides were then incubated with the human Whole Transcriptome Atlas (WTA) probe panel (Nanostring, NA-GMX-RNA-NGS-HuWTA-4, Lot HWTA21003) at 37°C in a humidified environment overnight. Sections were washed according to standard protocols and direct immunofluorescence was performed with antibodies against CD34 conjugated to Alexa-647 (Novus Biologicals, clone QBEnd-10, NBP2-34713AF647), Pan-cytokeratin conjugated to to Alexa532 (Novus Biologicals, clone AE1/AE3, NBP2-33200), CD45 conjugated to Alexa594 (clone 2B11 + PD7/26, Novus Biologicals, NBP2-34528), and a nuclear counterstain (Syto-13, Nanostring) DSP device run and library preparation were performed according to the manufacturers’ protocol. The library was sequenced using an Illumina NextSeq 550 sequencer with the specifications provided by Nanostring (paired-end reads at length 27, index length 8) at a library concentration of 1.6 pM using a NextSeq 550 High Output 75-cycle kit (Illumina, 20024906). Nuclei counts of regions of interest were obtained via the default automatic nuclear segmentation settings of the DSP device software.
2024-09-18 | GSE272995 | GEO
Project description:Objectives: Formalin-fixed paraffin-embedded (FFPE) tissue is the standard material for di-agnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction pro-tocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of func-tionally relevant proteins. Methods: Using the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was meas-ured by protein concentration and western blot analysis of cellular compart-ment markers as well as liquid chromatography-coupled mass spectrometry (LC-MS). Results: Commercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval. Conclusions: Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.
2022-05-19 | PXD029133 | Pride
Project description:Spatial tissue proteomics integrating whole-slide imaging, laser microdissection and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE® robotic system, which has the capacity to process 192 samples in three hours. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, particularly pertinent for laser microdissected workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell and epithelial microregions of 4,000 µm2 to a depth of ~2,000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.
2024-03-22 | PXD047762 | Pride
Project description:Gene expression profiling was performed on nasal scrape samples obtained from asthmatics and matched healthy controls, to identify markers associated with various asthma subtypes. Nasal scrape samples were collected from asthmatics and healthy controls and subjected to expression profiling using Affymetrix HG-U133Plus2.0 microarrays.
2015-10-23 | E-GEOD-41862 | biostudies-arrayexpress
Project description:The AE3 (Slc4a3) Cl/HCO3 exchanger is highly abundant in heart, but its function is not known. To assess its function we performed RNA Seq analysis of mRNA from hearts of AE3-null and wild-type mice. The patterns of differential gene expression indicate mild hypoxia, enhanced glucose metabolism, and reduced fatty acid metabolism in cardiac myocytes, which is consistent with the hypothesis that HCO3 extrusion by AE3 plays a role in carbon dioxide disposal.
2017-07-01 | GSE70471 | GEO
Project description:Lung, liver, muscle (Semitendinosus), small intestine (duodenum; approximately 5 cm from stomach) were collected from six cross-bred white pigs (three 15 week old and three 17 week old). Total RNAs were isolated from these tissues to study gene expression by using the first generation of porcine long oligonucleotide microarray. In the experimental design, tissue samples from the six animals were assigned to six loops (three for younger animals and three for older animals). One sample from each of the four tissues from a single animal was used in each loop. The order of the tissues in each loop was varied so that all pairs of tissues were represented together on a slide an equal number of times. Dye balance was used throughout so that each tissue was measured an equal number of times with each dye. Data from 48 measurements from 24 slides were collected, with 12 measurements for each tissue. The mixed model included fixed effects for dye, tissue, animal age, and age-by-tissue interaction as well as random effects for slide, animal, and animal-by-tissue interaction. Genes with expression significantly higher in one tissue than in each of the others will be referred to as tissue-selective genes. To identify such genes, pair-wise comparisons of normalized signal intensities between tissues were conducted as part of our mixed model analyses for each gene. Keywords: repeat sample
2005-03-05 | GSE2335 | GEO
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Project description:The goal of this study was to compare global RNA expression data obtained from: i) duodenal organoids cultured in conventional plastic-adherent Matrigel drop overlaid with growth medium; ii) Duodenum Intestine-Chips derived from organoids in the presence of constant flow and stretch; iii) human adult duodenum tissue and to verify whether Duodenum Intestine-Chip faithfully recapitulates human adult duodenum tissue and to better understand how much it differs from the organoids from which it’s derived.
2020-01-06 | GSE135196 | GEO
Project description:To assess gene expression underlying alcohol memory in different regions of the brain, we performed total RNA-sequencing (RNA-seq) on brain tissue (prefrontal cortex or dorsal hippocampus) from C57BL/6 adult mice that had undergone alcohol placement conditioning and either placed back into alcohol context (retrieval, Ret) or briefly handled as a control (no retrieval, NoRet)
2023-03-07 | GSE205586 | GEO
Project description:DNA methylation is tightly regulated throughout mammalian development and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CpG islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO induced AML. Using this model, we show that the primary effect of Tet2 loss in pre-leukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner, but increase relative to population doublings. We confirm this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many downregulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation, and that it is the combined silencing of several tumor suppressor genes in TET2-mutated hematopoietic cells that contribute to increased stem cell proliferation and leukemogenesis. Gene expression profiles (Agilent SurePrint G3 Mouse GE 8x60K arrays) of FACS-sorted in vivo GMP cells (Lin-cKit+Sca1-CD16/32+CD150-) isolated from bone marrow of recipient mice one month after transplantation of WT bone marrow or splenic cells from two independent moribond Tet2-/-:AE leukemic mice (LeuA/AE1), or LeuB/AE3)
2015-04-30 | E-GEOD-68415 | biostudies-arrayexpress