Abstract
BackgroundTranscriptome analysis of porcine whole blood has several applications, which include deciphering genetic mechanisms for host responses to viral infection and vaccination. The abundance of alpha- and beta-globin transcripts in blood, however, impedes the ability to cost-effectively detect transcripts of low abundance. Although protocols exist for reduction of globin transcripts from human and mouse/rat blood, preliminary work demonstrated these are not useful for porcine blood Globin Reduction (GR). Our objectives were to develop a porcine specific GR protocol and to evaluate the GR effects on gene discovery and sequence read coverage in RNA-sequencing (RNA-seq) experiments.ResultsA GR protocol for porcine blood samples was developed using RNase H with antisense oligonucleotides specifically targeting porcine hemoglobin alpha (HBA) and beta (HBB) mRNAs. Whole blood samples (n = 12) collected in Tempus tubes were used for evaluating the efficacy and effects of GR on RNA-seq. The HBA and HBB mRNA transcripts comprised an average of 46.1% of the mapped reads in pre-GR samples, but those reads reduced to an average of 8.9% in post-GR samples. Differential gene expression analysis showed that the expression level of 11,046 genes were increased, whereas 34 genes, excluding HBA and HBB, showed decreased expression after GR (FDR <0.05). An additional 815 genes were detected only in post-GR samples.ConclusionsOur porcine specific GR primers and protocol minimize the number of reads of globin transcripts in whole blood samples and provides increased coverage as well as accuracy and reproducibility of transcriptome analysis. Increased detection of low abundance mRNAs will ensure that studies relying on transcriptome analyses do not miss information that may be vital to the success of the study.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-954) contains supplementary material, which is available to authorized users.
Highlights
Transcriptome analysis of porcine whole blood has several applications, which include deciphering genetic mechanisms for host responses to viral infection and vaccination
Comparisons of globin reduction methods To determine the suitability of the Globin Reduction (GR) process for porcine whole blood samples, we initially evaluated the efficacies of three distinct methods (GLOBINclearTM, biotinylated Peptide Nucleic Acids (PNA) and RNase H) with whole blood samples drawn from 12 pigs collected in either PAXgeneTM (n = 3) or TempusTM (n = 9) tubes
The manufacturer confirmed that porcine hemoglobin alpha (HBA) and HBB sequences had low sequence homology to their corresponding human oligonucleotide probes used in the GLOBINclearTM-Human Kit, but the degree of dissimilarity is not known because the human probe sequences used in the GLOBINclearTM Kit are not publicly available
Summary
Transcriptome analysis of porcine whole blood has several applications, which include deciphering genetic mechanisms for host responses to viral infection and vaccination. Several globin RNA reduction protocols have been successfully applied to gene expression studies in human [6,7,8,9]. GLOBINclearTM (Ambion, Austin, TX, USA), a commercial product widely used in human clinical research, removes up to 95% of the HBA and HBB transcripts in human whole blood samples and improves the efficacy of gene expression assays [4,10,11]. (Thousand Oaks, CA, USA) [9,10] have differential reduction rates of globin transcripts in human blood. Globin RNA reduction improved the sensitivity and reproducibility of high throughput mRNA expression analysis of whole human blood samples [3,4,5,7,9,10]. There is, neither a commercial GR product available nor any literature demonstrating the efficiency and effects of GR at global level for porcine whole blood [2]
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