Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq.
Abstract
BACKGROUND: RNA-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. RESULTS: We 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.
CONCLUSIONS: Transcriptome 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.
Type
Journal articleSubject
Adipose Tissue, WhiteAnimals
Gene Expression Profiling
RNA
Rats
Sequence Analysis, RNA
Transcriptome
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https://hdl.handle.net/10161/15379Published Version (Please cite this version)
10.1186/s12896-015-0155-7Publication Info
Faherty, SL; Campbell, CR; Larsen, PL; & Yoder, AD (2015). Evaluating whole transcriptome amplification for gene profiling experiments using
RNA-Seq. BMC Biotechnol, 15. pp. 65. 10.1186/s12896-015-0155-7. Retrieved from https://hdl.handle.net/10161/15379.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Ryan Campbell
Postdoctoral Associate
Anne Daphne Yoder
Braxton Craven Distinguished Professor of Evolutionary Biology
My work integrates field inventory activities with molecular phylogenetic techniques
and geospatial analysis to investigate Madagascar, an area of the world that is biologically
complex, poorly understood, and urgently threatened. Madagascar has been designated
as one of the most critical geographic priorities for conservation action, retaining
less than 10% of the natural habitats that existed before human colonization. It is
critical that information be obtained as quickly as possible to docum
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