Abstract
BackgroundGene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples. However, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. We compared cDNA-mediated annealing, selection, and ligation (DASL)- and in vitro transcription (IVT)-based genome-wide expression profiling assays on RNA samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue.ResultsThe DASL-based platform produced expression results of greater reliability than the IVT-based platform in artificially degraded reference brain RNA and RNA from frozen tissue-based samples. Although data associated with a small sample of formalin-fixed RNA samples were poor when obtained from both assays, the DASL-based platform exhibited greater reliability in a subset of probes and samples.ConclusionsOur results suggest that the DASL-based gene expression-profiling platform may confer some advantages on mRNA assays of the brain over traditional IVT-based methods. We ultimately consider the implications of these results on investigations of neuropsychiatric disorders.
Highlights
Gene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples
To overcome the difficulties associated with poly A/oligo-dT-based priming in special experimental conditions such as with profiling partially degraded RNA, the DASL-based assay uses random priming at the cDNA synthesis step
We investigated the utility of the standard in vitro transcription (IVT)- and DASL- based genome-wide expression profiling assays in the context of a clinically important neuropsychiatric disorder, autism
Summary
Gene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples These methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. Gene expression profiling investigations involving postmortem brain tissue of cases with neuropsychiatric disorders such as autism have been limited due to tissue availability and tissue quality [1,2,3]. Such investigations, are critical for understanding uniquely human disorders [4]. The assay requires only a ~50 nucleotide target sequence for query oligonucleotide annealing, which makes it effective for quantifying partially degraded RNA samples
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