Polysomal and total RNA in controls and after 10 min global brain ischemia and 8 hours of reperfusion
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE82146 | GEO | 2016/06/02
SECONDARY ACCESSION(S): PRJNA324171
REPOSITORIES: GEO
Similar Datasets
Project description:Complete global brain ischemia (CGBI) and reperfusion occur following resuscitation from cardiac arrest. Different brain neurons are selectively vulnerable to CGBI: pyramidal neurons of hippocampal CA3 survive 10 min CGBI but those of CA1 die at 3 days following 10 min CGBI. CA3 neurons are expected to have more robust stress responses and repair responses than CA1 neurons. We used microarrays to compared total and polysome-bound mRNAs in CA1 and CA3 at 8 hr reperfusion after 10 min CGBI in Long Evans male rats to ascertain differences in total vs polysome-bound gene expression. Male Long Evans rats were subjected to (1) sham operation (non-ischemic control, NIC) or normothermic CGBI of 10 min followed by 8 hr reperfusion (8R). Hippocampal CA1 and CA3 were dissected. n = 5 CA1 or CA3 were pooled to give a single replicate and there were 3 or 4 replicates per group. Post-mitochondrial supernatant (PMS) was prepared. Twenty percent of PMS was TRIzol extracted to give total RNA. The remainder was run on a 20% sucrose pad to isolate polysome pellets, which were also TRIzol extracted to give polysome RNA. Total and polysome RNA were then run on Affymetrix Rat Gene 2.0 microarrays.
2016-06-02 | E-GEOD-82146 | biostudies-arrayexpress
Project description:We investigated mRNA regulatory proteins of the ELAV family following 10 min global brain ischemia and 8 hrs reperfusion (8R) or non-ischemic controls (NIC) in rat hippocampal CA1 and CA3. There were three main experiments: (1) Shot-gun proteomics of polysome pellets from NIC and 8R CA1 and CA3, (2) Shot-gun proteomics of ELAV immunoprecipitate eluents from NIC and 8R CA1 and CA3, and (3) Proteomics of ELAV antisera-reactive bands excised from nitrocellulose following ELAV Western blot.
2016-07-07 | PXD004078 | Pride
Project description:DO11.10 x TCR-Calpha-/- T cells stimulated under Th2 conditions for 6 days were centrifuged over Histopaque 1083 (Sigma) to remove dead cells, washed and rested for 20 min at 37C in complete media with 0.1 mg/ml cycloheximide (CHX, Sigma) added for the final 10 min. Restimulated cells were activated with 10 ug/ml plate-bound I-Ad/ova complexes for 3 hrs. Cells were washed with ice-cold PBS/CHX and resuspended in polysome extraction buffer [30 mM HEPES (pH 7.4), 15 mM MgCl2, 0.3 M NaCl, 1% Triton X-100, 0.1 mg/ml CHX, 1 mg/ml heparin, 500 U/ml SUPERase-In RNAse inhibitors (Ambion, Austin, TX), 1x Complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN)]. After incubation on ice for 25 min, nuclei and debris were removed by centrifuging and the cleared supernatants transferred to fresh tubes. 1.5 ml of each extract (from 3 x 10exp8 T cells) were layered on a 37.5 ml 10-50% sucrose gradient prepared in SW28 ultraclear centrifuge tubes (Beckman Coulter, Fullerton, CA) using the salts described above, but without detergent, RNAsin or protease inhibitors. After centrifugation for 220 min at 28,000 rpm (average RCF = 103,745), fractions were isolated using an ISCO density gradient fractionator (Lincoln, NE) by pumping 60% sucrose into the bottom of the tube and collecting the displaced volumes into acid phenol:chloroform, with constant monitoring of ultraviolet absorbance at 254 nm. Extracted RNA was precipitated with isopropanol, washed and resuspended in distilled water. RNA from polysome associated fractions 7-12 of the gradients described above were pooled and 30-100 ug of total RNA per sample were used to template a Superscript III reverse transcription reaction (Invitrogen, Carlsbad, CA) incorporating amino-allyl dUTP (Sigma). cDNA samples were washed with distilled water on microcon-30 columns (Millipore, Billerica, MA), dried and labeled with Cy3 (primed samples) or Cy5 (restimulated samples) using CyDye reactive dye labeling kits (Amersham). Unincorporated dye was removed using QiaQuick PCR purification columns (Qiagen), labeled cDNAs were eluted in 10mM Tris, pH 8, combined and dried. Fluorescence in the Cy3 and Cy5 channels was acquired using an Axon 4000B microarray scanner and GenePix software (Axon Instruments, Union City, CA). Data were normalized using the R package Bioconductor software and lowess normalization on the pixel medians without background subtraction (Ihaka and Gentleman, 1996). Keywords = Th2 differentiation Keywords = translation Keywords = polysome profile Keywords: cell stimulation
2006-03-23 | GSE4500 | GEO
Project description:N6-Methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the stress response in the adult brain in vivo are currently unknown. Here, we investigated the effect of gene deletion of Mettl3, a m6A methyltransferase, and Fto, a m6A and m6Am demethlyase, induced in adulthood in excitatory neurons of the CA1 and CA3 in the hippocampus (Nex-CreERT2 Mettl3 or Fto cKO) on the transcriptome of CA1 and CA3 as well as the transcriptomic response of the CA1 and CA3 transcriptome to fear conditioning.
2018-07-10 | GSE113796 | GEO
Project description:Neurodegenerative brain disorders become more common in the aged. Most of these disorders are associated with or caused by selective death of certain neuronal subpopulations. The mechanisms underlying the differential vulnerability of certain neuronal populations are still largely unexplored and few neuroprotective treatments are available to date. Elucidation of these mechanisms may lead to a greater understanding of the pathogenesis and treatment of neurodegenerative diseases. Moreover, preconditioning by a short seizure confers neuroprotection following a subsequent prolonged seizure. Our goal is to identify pathways that confer vulnerability and resistance to neurotoxic conditions by comparing the basal and preconditioned gene expression profiles of three differentially vulnerable hippocampal neuron populations. Hippocampal CA1 and CA3 pyramidal neurons are highly susceptible to seizures and ischemia, whereas dentate gyrus granule cells are relatively resistant. A brief preconditioning seizure confers protection to the pyramidal cells. We will first determine gene expression profiles of untreated rat CA1 and CA3 pyramidal cells, and dentate granule cells, using laser capture microscopy to obtain region-specific neuronal mRNA. We will then determine the effect of a brief preconditioning seizure, which is neuroprotective in CA1 and CA3, on these expression profiles. We hypothesize that common molecular mechanisms exist in neurons that determine their susceptibility to seizure-induced injury. Intrinsic differences in gene expression exist between hippocampal glutamatergic CA1 and CA3 pyramidal neurons, on the one hand, and dentate granule cells on the other, which contribute to the greater susceptibility of pyramidal neurons to degeneration in experimental stroke and epilepsy. We specifically hypothesize that differences in basal energy metabolism genes may confer differential susceptibility to neurodegeneration produced by seizures and ischemia. Anesthetized animals will be sacrificed by decapitation, and frozen 10 micron sections will be lightly stained with cresyl violet to identify cell layers in the hippocampus. Approximately 1000 neurons from each of the three cell layers will be isolated by LCM. Poly-A RNA will be amplified using a modified Eberwine protocol. The quality of our aRNA will be evaluated by quantitative RT-PCR of GluR6 and KA2 mRNA levels before we send the samples to the Center for labeling and hybridization to Affymetrix rat 230A arrays. We will provide a one-round amplification cDNA product to the center for labeling and hybridization. This protocol is identical to a previously approved study by Jim Greene in our laboratory.
2007-09-18 | E-GEOD-1994 | biostudies-arrayexpress
Project description:Study on selective vulnerability of certain brain regions to oxidative stress. Here we selected 4 brain regions (hippocampal CA1 and CA3, cerebral cortex, and cerebellar granular layer) to study this phenomenon. Experiment Overall Design: Neurons were collected from the 4 regions of the rat brain and subjected to Affymetrix RAE230A analysis, in order to identify genes related to the differential vulnerability of the neurons to oxidative stress.
2008-04-07 | E-GEOD-7139 | biostudies-arrayexpress
Project description:Neurodegenerative brain disorders become more common in the aged. Most of these disorders are associated with or caused by selective death of certain neuronal subpopulations. The mechanisms underlying the differential vulnerability of certain neuronal populations are still largely unexplored and few neuroprotective treatments are available to date. Elucidation of these mechanisms may lead to a greater understanding of the pathogenesis and treatment of neurodegenerative diseases. Moreover, preconditioning by a short seizure confers neuroprotection following a subsequent prolonged seizure. Our goal is to identify pathways that confer vulnerability and resistance to neurotoxic conditions by comparing the basal and preconditioned gene expression profiles of three differentially vulnerable hippocampal neuron populations. Hippocampal CA1 and CA3 pyramidal neurons are highly susceptible to seizures and ischemia, whereas dentate gyrus granule cells are relatively resistant. A brief preconditioning seizure confers protection to the pyramidal cells. We will first determine gene expression profiles of untreated rat CA1 and CA3 pyramidal cells, and dentate granule cells, using laser capture microscopy to obtain region-specific neuronal mRNA. We will then determine the effect of a brief preconditioning seizure, which is neuroprotective in CA1 and CA3, on these expression profiles. We hypothesize that common molecular mechanisms exist in neurons that determine their susceptibility to seizure-induced injury. Intrinsic differences in gene expression exist between hippocampal glutamatergic CA1 and CA3 pyramidal neurons, on the one hand, and dentate granule cells on the other, which contribute to the greater susceptibility of pyramidal neurons to degeneration in experimental stroke and epilepsy. We specifically hypothesize that differences in basal energy metabolism genes may confer differential susceptibility to neurodegeneration produced by seizures and ischemia. Anesthetized animals will be sacrificed by decapitation, and frozen 10 micron sections will be lightly stained with cresyl violet to identify cell layers in the hippocampus. Approximately 1000 neurons from each of the three cell layers will be isolated by LCM. Poly-A RNA will be amplified using a modified Eberwine protocol. The quality of our aRNA will be evaluated by quantitative RT-PCR of GluR6 and KA2 mRNA levels before we send the samples to the Center for labeling and hybridization to Affymetrix rat 230A arrays. We will provide a one-round amplification cDNA product to the center for labeling and hybridization. This protocol is identical to a previously approved study by Jim Greene in our laboratory. Keywords: other
2004-11-24 | GSE1994 | GEO
Project description:The hippocampal formation is a brain structure essential for higher-order cognitive functions. It has exquisite differences in anatomical organization and cellular composition, and hippocampal sub-regions have different properties and functional roles. Areas CA1 and CA3 in particular, are key sub-regions for learning and memory formation that fulfill complementary but specific functions. The molecular basis for such specific properties and the link to learning and memory remain unknown. Here using a SWATH-MS proteomic approach and bioinformatic tools, we identify a selective proteomic signature in area CA1 and CA3, and reveal their specific dynamics during memory formation. We show that 30% of all quantifiable proteins are differentially expressed in area CA1 and CA3 at baseline, and that each proteome responds differently during the formation of memory for object or object location. Using clustering and cross-correlational analyses, we outline specific temporal proteomic profiles and an increased correlation between both forms of memory within area CA1, but not within area CA3. These results provide new insight into a proteomic basis for hippocampal sub-region molecular and functional specificity.
2018-03-28 | PXD006382 | Pride
Project description:KDM5A is a chromatin remodeler disrupted in neurodevelopmental disease. To investigate the effect of losing KDM5A on the development of the hippocampus, we profiled hippocampi from wild type (WT) and Kdm5a -/- mice using single-nuclei RNA sequencing (snRNA-seq). We found that KDM5A regulates the development of specific subtypes of excitatory (CA1, CA2, CA3) and inhibitory (SST+, PVALB+) neurons, that loss of KDM5A leads to a more mature cellular identity in the hippocampus, and that KDM5A functions early in development to specify proper CA1 cell identity.
2023-09-01 | GSE235490 | GEO
Project description:The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO, underscore the relevance of GRTH in translation. Studies showed the association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. Microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice.
2012-11-21 | E-GEOD-33860 | biostudies-arrayexpress