Gene expression in tolerant and non-tolerant liver allografts
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE175397 | GEO | 2022/02/09
REPOSITORIES: GEO
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Project description:A control group consisting of nine C57/BL/6J mice was maintained on a normal diet consisting of Purina Rodent Chow (Harlan Teklad, catalog #5008, 6.5% fat, 49% carbohydrate, 23% protein, 3.5 kcal/ g). The experimental group consisted of nine mice grown on a specialty high-fat diet (Harlan Teklad, catalog #TD88137, 42.16% fat, 42.81% carbohydrate, 15.02% protein, 4.53 kcal/ g). Mice were maintained for ten weeks on their respective diets in a controlled environment with standard light and feeding schedule. Two days before tissue harvest, the experimental group on the high-fat diet was divided into one group of five and one group of four mice. The first group continued on the high-fat diet for the final two days, while the second group fasted. Mouse weights were recorded two days prior, and the day of tissue harvest. At the end of the fasting period liver tissue samples were harvested from each mouse. RNA was purified from the liver tissue samples. Each tissue was homogenized in 1 mL of STAT-60 (Tel-Test, Inc., Friendswood, TX) and the homogenate was mixed with 200 uL of chloroform. The aqueous phase was removed, and mixed with 500 uL of isopropanol to precipitate the RNA. The resulting RNA pellet was washed with 500 uL of 70% ethanol and allowed to air dry. The pellet was dissolved in RNase free water (Ambion, TX), quantified by UV spectroscopy, and stored at -80C. Total RNA control samples (Universal Mouse Reference RNA, catalog # 740100, Stratagene) were labeled using Cy3 dCTP (Perkin-Elmer), and liver total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). The control RNA samples were additionally mixed with 2 uL of random hexamers. The reaction mixtures were incubated at 42 C for two hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 1 N NaOH and incubation at 65 C for 10 minutes. The alkaline conditions were neutralized by addition of 2 uL of 1 N HCl. Liver cDNA samples were then mixed with a control cDNA sample, and the mixtures were cleaned using a QIAquick Nucleotide Removal kit (Qiagen) as described by the manufacturer's instructions. The cleaned samples were concentrated in a speed vacufuge (Eppendorf) for 20 minutes at 65 C, and were dissolved in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), quantified, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 C using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), then in 1X SSC, and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.). Microarrays were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). Arrays contained 16,847 features, printed from a synthesized oligonucleotide mouse library (Operon). The library was suspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed at approximately 50% humidity. Once printed, the probes were crosslinked using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride (Fluka AG), 239 mL 1-methyl-2-pyrrolidinone (EM Science), and 10.7 mL of 1 M boric acid (Fisher), pH 8.0. They were cleaned by rinsing twice in 250 mL of milli-Q water, followed by 250 mL of 95% ethanol (Pharmco Products). After blocking, they were dried using filtered air and stored in the dark at room temperature. In this experiment the gene expression data of the control mice is given by samples GSM6592, GSM6593, GSM6594, and GSM6595. The gene expression data of the high-fat mice is given by samples GSM6599, GSM6600, GSM6601, and GSM6602. The gene expression data of the fasted mice is given by samples GSM6596, GSM6597, and GSM6598. Keywords = mouse, liver, energy homeostasis, gene expression, microarrays Keywords: repeat sample
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2009-11-10 | GSE18837 | GEO
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
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2012-01-09 | E-GEOD-26625 | biostudies-arrayexpress
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