Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of hearts and kidneys of spontaneously hypertensive rat and coisogenic rat strain with connexin50 mutation

ABSTRACT: We aimed to compare transcriptome in heart and livers of spontaneously hypertensive rat SHR/OlaIpcv (Rat Genome Database ID: 631848) versus coisogenic rat strain SHR-Gja8m1Cub (Rat Genome Database ID: 2293729) using Affymetrix GeneChipÂ® Rat Exon 1.0 ST Arrays in triplicate. Affymetrix GeneChipÂ® Rat Exon 1.0 ST Array was used to determine the transcriptomic characteristics of the SHR and the coisogenic strain SHR-Dca. We extracted total RNA from the kidneys and hearts of 4-month old males of both strains using phenol-chloroform and purified with the RNeasy Mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturerâs protocol. The quality of the total RNA was verified by the Agilent 2100 Bioanalyzer system. Double-stranded complementary DNA (cDNA) was synthesized from total RNA. The labeled and fragmented cDNA was pooled for each strain (SHR, SHR-Dca) and tissue (heart, kidney) and hybridized to Affymetrix GeneChipÂ® Rat Exon 1.0 ST Arrays in triplicate (total 12 arrays). The whole hybridization procedure including DNA adjustment was performed according to the protocol recommended by Affymetrix.

ORGANISM(S): Rattus norvegicus

SUBMITTER: OndÅej Âeda

PROVIDER: E-MTAB-4542 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The study sought to investigate differences in onset and progression of renal disease in the Dahl salt-sensitive (S), S.SHR(2) congenic, and spontaneously hypertensive rat (SHR) using a time-course. The data clearly demonstrates that the locus on chromosome 2 has a major and sustained ability to attenuate renal damage. As early as week 4, significant interstitial changes were observed between the S and the congenic which preceded any significant difference in proteinuria. Gene expression profiling was performed at week 4, 12, and 20 using kidney from the S and S.SHR(2) congenic to: (1) identify expression differences of positional candidates within the QTL region; (2) correlate temporal gene expression changes between the S and congenic with degree of renal damage; and (3) identify biochemical pathways potentially involved in the attenuated renal damaged observed in the congenic. Gene pathway analysis (IngenuityÂ® Systems) performed at week 4, 12, and 20 revealed that pathways involved in cellular assembly and organization, cellular movement, and immune response were controlled differently between the S and congenic. Considering all the data, the chromosome 2 congenic appears to attenuate renal damage primarily through an altered fibrotic response. Experiment Overall Design: DNA microarray analysis was performed using Affymetrix GenechipÂ® Rat Genome 230 2.0 array at three timepoints. The chip contains 31,000 probe sets from more than 30,000 transcripts on one array. Three male S and three male S.SHR(2) congenic were selected at random from each group test for proteinruia at week 4, 12 and 20. Kidney was cut into ~ 0.5cM size cubes, suspended in RNAlater (Ambion, Austin, TX) and stored overnight at 4ÂºC. RNA was extracted using TrizolÂ® reagent (Invitrogen, Carlsbad, CA) and purified using Mini RNeasy kit (Qiagen,Valencia, CA ) according to manufacturerâs protocols. RNA quality was assessed by an OD260/280 ratio > 2.0 and visually by ethidium bromide staining on an agarose gel. Experiment Overall Design: Biotinylated cRNA was synthesized from 10 ug of total RNA using the One-Cycle Target Labeling Kit (Affymetrix, Santa Clara, CA) as directed by the user manual. cRNA quality for each sample was assessed by hybridization of 5ug adjusted kidney cRNA to Affymetrix GenechipÂ® Test3 array. Subsequently, 15ug adjusted kidney cRNA was hybridized to the GenechipÂ® Rat 230 2.0 array. Hybridized chips were automatically washed, stained and scanned at the Medical University of Ohio Bioinformatics & Proteomics/Genomics Program gene array facility using Affymetrix equipment.

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Transcription profiling of hearts and kidneys of spontaneously hypertensive rat and coisogenic rat strain with connexin50 mutation

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