Real-time quantitative PCR analysis of human ependymal and ventral horn regions in adult control spinal cord
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE65463 | GEO | 2015/04/12
SECONDARY ACCESSION(S): PRJNA274061
REPOSITORIES: GEO
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Project description:Adult human ependymal and ventral horn regions were obtained from postmortem frozen samples by Laser Capture Microdissection. Briefly, Cryostat 25 micron sections from were stained with toluidin blue and both regions microdissected and collected on eppendorf (n=4 for each region). Samples mRNA concentration and purity was assessed by electrophoresis (BioRad Experion HighSensitivity kit, USA). RQI values were lower than 6,5 in every case, so that purification was followed by 2 cycle amplification with a kit designed for highly degraded samples (ExpressArt® TRinucleotide mRNA Amplification Kit; #6299-A15, AmpTec, AMSBIO, UK). After amplification, mRNA concentration and purity was assessed both by electrophoresis (BioRad Experion StSens kit, USA) and by spectrophotometry (Nanodrop, Thermo Scientific, USA). We amplified 3.7-37 ng of total RNA, obtaining between 6 and 21 µg of mRNA after 2 rounds. After collecting samples and studying the RNA integrity and quantity, cDNA of samples was selected for gene expression assays using 384 wells Custom Taqman Low Density Arrays. We built arrays with genes belonging to a profile of stemness or ependymoma (see Garcia-Ovejero et al., 2015, BRAIN). Taqman based qPCR gene expression profiling. Ependymal and ventral horn regions obtained by LCMD from four different individuals each were used to establish genes involved in stem cell niches or in ependymoma phenotype that are enriched in control human ependyma using ventral horn as a non-ependymary, non-neurogenic region. Samples were treated as stated in the summary. Equal amount of amplified RNA (aRNA; 25ng, corresponding approximately to 500ng total RNA) from each donor was used in Custom Designed Taqman Low Density Arrays. Every value is the resultant of duplicates at least, but most of them have been assayed 4 times.
2015-04-12 | E-GEOD-65463 | biostudies-arrayexpress
Project description:The main aim of the study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgeneTM and TempusTM tubes. Comparisons of present call rates, variances, correlations and influence of globin reduction across the two collection systems was performed using in vivo glucocorticoid stimulation in 24 peripheral blood samples from three individuals. Three healthy male volunteers aged 30-35 years with body mass index of 20-25 were enrolled in the study.1.5 mg dexamethasone stimulated peripheral blood samples were drawn 3 hours later. For each sample, duplicate measures of 2.5 ml and 3ml blood was collected in PAXgeneTM tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and TempusTM tubes (Applied Biosystems, Foster City, CA, USA) respectively. The PAXgeneTM and TempusTM tubes were stored for 2.5 hours at room temperature and then transferred to -20°C. Due to the lower RNA yield from the PAXgeneTM tubes, RNA from two PAXgeneTM tubes was pooled together for the experiment . Total RNA was isolated from whole blood stored in PAXgeneTM and TempusTM tubes according to the respective manufacturer's instructions. The PAXgeneTM samples were processed using the PAXgeneTM Blood RNA Kit based on the Quiagen method for column purification of nucleic acids (PreAnalytiX GmbH, Hombrechtikon, Switzerland, catalogue number 762174). The TempusTM samples were isolated using the TempusTM Spin RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA, catalogue number 4380204). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The concentration and purity of total RNA was independently assessed by 260/280 UV absorption ratios, respectively (Nanophotometer, Implen, Munich, Germany). RNA samples were divided into non-globin reduced and globin reduced groups. The GLOBINclearTM-Human Kit (Ambion, Inc., Texas, USA, catalogue number #AM1980) was used to remove globin mRNA. Sample amplification and labeling for both globin-reduced and non-reduced samples was performed using the IlluminaR TotalPrep RNA Amplification Kit (Ambion, Inc., Texas, USA, catalogue number AMIL1791).
2012-01-15 | GSE34039 | GEO
Project description:Illumina technology was used to generate mRNA profiles of stem apex of Populus yunnanensis with cutting and inverted cutting. Total RNA was extracted separately from each plant and pooled to three biological replicates per condition. RNA concentration and purity was measured using NanoDrop 2000(Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
2023-12-08 | GSE249347 | GEO
Project description:To identified the differential methylation of genes and regions to elucidate the mechanisms of toxicity related to environmental chromium exposure. Fasting whole blood samples were collected by clinicians in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, and stored them at −80 °C. A DNeasy Blood and Tissue Kit (Qiagen) was used to isolate DNA from whole blood. The purity and concentration of DNA were estimated using Nanodrop 2000 (ThermoScietific). Approximately 500 ng of genomic DNA from each sample was used for sodium bisulfite conversion with the EZ DNA methylation Gold Kit (Zymo Research, USA) in accordance with the manufacturer’s standard protocol. Genome-wide DNA methylation was performed using the Illumina Infinium HumanMethylation850K (EPIC) BeadChip (Illumina Inc, USA) in accordance with the manufacturer’s instructions.
2022-12-13 | GSE206418 | GEO
Project description:Gene expression microarray was performed using early log phase culture at 37 degree and 200 rpm followed by RNA extraction from M.tb H37Rv, HigB KO M.tb H37Rv and complemented Hig B KO M.tb H37Rv For gene expression profiling, total RNA was isolated from wild type H37Rv, higB KO mutant strain and complemented strains. The bacterial pellets for M.tb H37Rv wild type, M.tb H37Rv HigB KO mutant strain and complemented strain were resuspended in 1.0 ml TRIzol (Invitrogen Corporation, Carlsbad, CA); mRNA was extracted as per standard protocols and cleaned using RNAeasy columns (Qiagen GmbH, Hilden, Germany). Extracted mRNA (1 μg) was treated with DNase I using the Ambion DNA-free kit (Invitrogen Corporation, Carlsbad, CA) to remove genomic DNA contamination and quantified using Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). The purity and integrity of RNA samples were assessed Agilent 2100 Bio analyzer (Agilent TechnologiesInc., USA). Further, 25ng of RNA was amplified and labeled using Low input Quick Amp WT Labeling kit (Agilent Technologies, USA). The labeled cRNA was purified using RNAeasy columns (Qiagen, USA) and total yields were quantified using Nanodrop 2000c spectrophotometer.
2021-11-30 | GSE179403 | GEO
Project description:For the bilateral intrastriatal delivery of AAV expressing control or TRAX shRNA, mice were anesthetized with ketamine/xylazine HCl (87.56 and 7.5 ug/kg, respectively, via an intraperitoneal injection) and injected with the indicated AAV (2 µl per injection spot, 2.5*10^9 vector genome/µl) with a 10 ml syringe (Hamilton, Reno, NV, USA) at a rate of 0.5 ul/min. The AAV was injected at the desired position: AP+0.5, L±2, DV-2.5 and -3.5 mm relative to bregma and the dura surface. Then the mouse brains were removed from the mice directly for dissection of striatum. RNA was extracted by TRIzol reagent or miRNeasy mini kit (Qiagen). DNase digestion was performed by the DNA-free™ Kit (Life Technologies, Carlsbad, CA, USA) or the RNase-free DNase set (Qiagen) following the manufacturer’s instructions. The concentration and purity of RNA were measured at 260, 280 and 230 nm by a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). RNA was qualified by a Bioanalyzer 2100 (Agilent Technology, Santa Clara, CA, USA) with an RNA 6000 LabChip kit (Agilent Technology). For mRNA, the RNA was prepared based on Illumina’s official protocol. Library construction was carried out by TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, San Diego, CA, USA) followed by AMPure XP beads (Beckman Coulter, Brea, CA, USA) size selection. Libraries were sequenced using sequencing-by-synthesis (SBS) technology (Illumina). Sequencing data and FASTQ reads were generated using an inhouse Welgene ’Biotech pipeline according to Illumina’s base calling program bcl2fastq v2.20.
2024-10-14 | GSE187608 | GEO
Project description:The mammalian oocyte has the unique feature of supporting fertilization and normal development while being able of reprogramming the nuclei of somatic cells towards pluripotency and occasionally even totipotency. Whilst oocyte quality is known to decay with somatic aging it is not a given that different biological functions decay concurrently. In this study we tested whether oocyte reprogramming ability decreases with somatic aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT) at levels similar to ooplasts of young donors. Despite differences in transcriptome between ooplasts of old and young mice gene expression profiles of the resultant SCNT blastocysts were very similar. Furthermore albeit results showing global down-regulation of apoptosis-related genes in climacteric ooplasts and the reported beneficial effect of inhibiting p53 on transcription factor-induced (iPS) pluripotency reprogramming capacity of young ooplasts was not affected by specifically blocking p53. Our observations strongly suggest that the outcome of oocyte-induced reprogramming is determined by the availability of intrinsic reprogramming factors tightly regulated throughout aging as well as intracellular and environmental checkpoints during pre-implantation development towards selection of the successfully reprogrammed embryos. While oocytes are not regenerated but rather last for life we further propose that these cells can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction. Total mRNA was isolated for samples from young and climacteric mice (pools of 20 ooplasts and 20 SCNT blastocysts in duplicate) using a RNeasy Mini Kit (Qiagen Valencia CA USA) as described by the manufacturer (no DNase treatment). RNA concentrations as well as purity and integrity check were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit (Agilent Technologies Palo Alto CA USA). RiboAmp HS Plus Amplification Kit (MDS Analytical Technologies Ismaning Germany) was used to amplify the total mRNA with two rounds of amplification according to the manufacturerâ??s instructions. Amplified RNA was eluted with 15 µL RE Buffer included in the RiboAmp HS Plus Amplification Kit. RNA concentrations were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit. Turbo Labeling CY3 Kit (MDS Analytical Technologies) was used to label 3 µg of amplified RNA with Cy3. Concentration and frequency of incorporation were measured with NanoPhotometer (Implen). Microarray wash and detection of the labeled RNA on GeneChips were carried out according to the manufacturerâ??s instructions (Agilent Technologies). Gene expression profiling was performed using Agilent Whole Mouse Genome Oligo Microarrays (4 x 44k each array containing 41174 features). Array image acquisition and feature extraction was performed using Agilent G2505B Microarray Scanner and Feature Extraction software v.9.5 (Agilent Technologies).
2011-08-16 | GSE23654 | GEO
Project description:The main aim of the study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgeneTM and TempusTM tubes. Comparisons of present call rates, variances, correlations and influence of globin reduction across the two collection systems was performed using in vivo glucocorticoid stimulation in 24 peripheral blood samples from three individuals. Three healthy male volunteers aged 30-35 years with body mass index of 20-25 were enrolled in the study.1.5 mg dexamethasone stimulated peripheral blood samples were drawn 3 hours later. For each sample, duplicate measures of 2.5 ml and 3ml blood was collected in PAXgeneTM tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and TempusTM tubes (Applied Biosystems, Foster City, CA, USA) respectively. The PAXgeneTM and TempusTM tubes were stored for 2.5 hours at room temperature and then transferred to -20°C. Due to the lower RNA yield from the PAXgeneTM tubes, RNA from two PAXgeneTM tubes was pooled together for the experiment . Total RNA was isolated from whole blood stored in PAXgeneTM and TempusTM tubes according to the respective manufacturer's instructions. The PAXgeneTM samples were processed using the PAXgeneTM Blood RNA Kit based on the Quiagen method for column purification of nucleic acids (PreAnalytiX GmbH, Hombrechtikon, Switzerland, catalogue number 762174). The TempusTM samples were isolated using the TempusTM Spin RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA, catalogue number 4380204). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The concentration and purity of total RNA was independently assessed by 260/280 UV absorption ratios, respectively (Nanophotometer, Implen, Munich, Germany). RNA samples were divided into non-globin reduced and globin reduced groups. The GLOBINclearTM-Human Kit (Ambion, Inc., Texas, USA, catalogue number #AM1980) was used to remove globin mRNA. Sample amplification and labeling for both globin-reduced and non-reduced samples was performed using the IlluminaR TotalPrep RNA Amplification Kit (Ambion, Inc., Texas, USA, catalogue number AMIL1791). RNA quality, yield and numbers of detected transcripts from the two RNA collection systems was comparable, with no significant differences between the tube types. Globin reduction resulted in a significant increase in present call rates in PAXgeneTM and TempusTM tubes and significant decrease in gene expression variance in both RNA collection tubes . Comparisons of glucocorticoid receptor-stimulated gene expression profiles between the two collection tube systems revealed an overlap of only 17 to 54%, depending on the stringency level of the statistical thresholds. This overlap increased by 1-8% when the RNA samples were processed to remove the globin mRNA. These results indicate that gene expression profiles obtained from PAXgeneTM and TempusTM differ drastically and should not be analyzed together. These data suggest that researchers must exert caution while interpreting expression profiles obtained through different RNA collection tubes.
2012-01-15 | E-GEOD-34039 | biostudies-arrayexpress
Project description:Klebsiella pneumoniae x546,ATCC15380, TMT,The samples were ground into a powder in liquid nitrogen. Then the powder was suspended in lysis buffer [1% sodium deoxycholate (SDS), 8M urea]. The mixture was allowed to settle at 4 鎺矯 for 30 min during which the sample were vortexed at every 5 min, and treated by ultrasound at 40 kHz and 40 W for 2 min. After centrifugation at 16000 rpm at 4鎺矯 for 30 min, the concentration of protein supernatant was determined by Bicinchoninic acid (BCA) method by BCA Protein Assay Kit (Pierce, Thermo, USA). Protein quantification was performed according to the kit protocol(Chen et al., 2021)
2021-11-15 | PXD028396 | Pride
Project description:T7-based linear amplification of low concentration mRNA samples using beads and microfluidics
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