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LECs from control and cirrhotic rats

ABSTRACT: The study was performed in liver-endothelial cells (LECs) isolated from 6 adult male Wistar rats, undergoing a cirrhosis induction protocol as described previously by our group, and LECs isolated from 6 control Wistar rats, according to the criteria of the Investigation and Ethics Committee of the Hospital Clínic Universitari (Spain).The aim of this study was to evaluate differences in the gene expression pattern between LECs from control and cirrhotic rats. Methods: LECs were isolated from livers by collagenase perfusion, isopycnic centrifugation and incubation of cells with magnetic beads coated with specific antibodies (Ab).Total RNA was extracted from LECs with Trizol reagent (Life Technologies, Rockville, MD) after the cell-isolation step described above. RNA quality and concentration were confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). cRNA samples were hybridized to the Rat Expression 230A oligonucleotide microarray (Affymetrix). Microarray samples preparation and processing procedures where performed at the IDIBAPS Genomic Unit (Hospital Clinic Universitari, Barcelona) following the protocol as described in the Affymetrix GeneChip Expression Analysis Manual (Affymetrix, Inc, Santa Clara, CA). Keywords: repeat sample

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE1843 | GEO | 2005/11/17

SECONDARY ACCESSION(S): PRJNA90455

REPOSITORIES: GEO

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Project description:One µg of total RNA from either an individual rat or from a pooled sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol. The amount and quality of the resulting fluorescently labeled cRNA was assessed using a Nanodrop ND-100 spectrophometer and an Agilent Bioanalyzer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the Agilent Rat Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17 hrs, prior to washing and scanning. Data was extracted from the resulting images using Agilent’s Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, CA). For day/night comparisons, two types of complementing hybridizations were performed: hepatic RNA from individual day rats was hybridized against a pool made up of RNA from 12 hour offset night rats, and RNA from individual night rats was hybridized against a pool made up of RNA from 12-hour offset day rats. Specifically, hepatic RNA samples from three individual day rats collected ten hours after light on (CT10) were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six night rats collected ten hrs after lights off (CT22); and RNA samples from three individual night rats collected at CT22 were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six day rats collected at CT10. This was replicated in a second study for a total of 24 hybridizations of 12 hour offset samples. For time of day comparisons, equal aliquots of RNA from the livers of six rats collected at each of four different times of the circadian day (CT4, CT10, C16 and CT22) were pooled. The two replicate studies thus resulted in 8 pools (two replicate pools/time point times four time points); each pool was hybridized against a Universal Rat Reference RNA Standard (Stratagene, La Jolla, CA). Keywords: time-course

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LECs from control and cirrhotic rats

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