Computational protocol: RNA SEQ Analysis Indicates that the AE3 Cl−/HCO3− Exchanger Contributes to Active Transport-Mediated CO2 Disposal in Heart

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Protocol publication

[…] Total RNA was isolated from whole hearts of 4-month-old FVB/N WT and AE3-null male mice (n = 4 of each genotype). All procedures using animals conformed to guidelines published by the National Institutes of Health (Guide for the Care and Use of Laboratory Animals) and were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. Samples were subjected to RNA Seq analysis in the University of Cincinnati Genomics and Sequencing Core using the Illumina HiSeq. 1000 platform. Sequence reads were aligned to the reference mouse genome using TopHat aligner. The full data set was deposited in the Gene Expression Omnibus (GEO accession number GSE70471) and is also provided as an Excel File in Supplementary Table . Additional Excel Files with subsets of expression data are provided in Supplementary Tables –. Fold-change expression data in the Excel files relate expression in AE3-null hearts relative to that observed in WT hearts.Statistical analysis to identify differentially expressed genes was performed using the negative-binomial model of read counts as implemented in DESeq Biocondoctor package. Most of the genes (80%) included in the figures had an FDR (False Discovery Rate) < 0.05, which is a measure of the probability of a false positive given the inclusion of over 23,000 genes in the analysis. Among the 536 genes with FDR < 0.05, 284 were up-regulated and 252 were down-regulated. Some genes with FDR > 0.05 were also included because they were in significantly enriched GO categories that were a major part of the phenotype (see Table and legends for Supplementary Tables –). Inclusion of genes with a significant P value but an FDR > 0.05 that were also present in significant GO categories, which were derived from a small subset of genes, protected against exclusion of false negatives. mRNA expression is presented as Reads Per Kilobase per Million mapped reads (RPKM), which normalizes for the size of the mRNA and provides a measure of the relative abundance of specific transcripts. Approximately 25 million reads were achieved per sample.Cardiac RNA Seq expression data for mammalian hearts (Supplementary Table ) and other tissues was obtained from the European Bioinformatics Institute (EBI) Expression Atlas (http://www.ebi.ac.uk/gxa/home). Heart data was for mRNA expression in hearts of both male and female Fisher 344 rats at 4 developmental stages and in hearts of C57Bl6 mice, opossum, rhesus monkey, and human. The latter study used a normalization procedure that allowed cross-species comparisons of specific genes. […]

Pipeline specifications

Software tools TopHat, DESeq
Databases GEO PCAWG
Application RNA-seq analysis
Organisms Mus musculus
Diseases Heart Failure, Glucose Intolerance
Chemicals Carbon Dioxide, Superoxides