Abstract
BackgroundRNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN’s Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates.ResultsWe found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison.ConclusionsTranscriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0155-7) contains supplementary material, which is available to authorized users.
Highlights
RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype
We aim to investigate the degree to which NuGEN’s Ovation RNA-Seq V2 system influences gene expression profiles in white adipose tissue excised from standard laboratory rats (Rattus norvegicus), building upon and enhancing knowledge regarding current whole transcriptome amplification (WTA)
We identified strong correlations in gene expression within each of the six pairwise comparisons of matched raw RNA and amplified RNA, and FPKM values averaged slightly higher in libraries constructed from raw RNA samples (Figure 1)
Summary
RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. RNA-Seq is quickly becoming the preferred method for comprehensively characterizing global transcriptome activity This approach has emerged as a powerful tool for determining the link between genotype and phenotype given that the transcriptomes of specific tissue types and individual cells reflect functionality [1,2,3,4]. When conducting RNA-Seq studies, one potential obstacle encountered by researchers can result from suboptimal starting quantities of RNA that the use of NGS platforms often requires This can be especially problematic for certain tissue types wherein starting RNA is in low quantities, such a white adipose tissue, or when using minimally invasive techniques to acquire samples [7,8,9,10]. Many of these approaches yield considerably less than a microgram of total RNA [11,12,13,14,15,16] though current library preparation protocols are typically optimized for a minimum of one microgram of total RNA
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