Abstract
BackgroundThe template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo.ResultsIn this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background.ConclusionsOur results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection.
Highlights
The template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts
Our results demonstrate that application of the improved TS-PCR method dramatically reduced the proportion of oligo concatamers in the resulting sequence data, and the cDNAs produced were of high quality and yield, useful for both conventional Sanger sequencing as well as next-generation sequencing
Based on results presented below, we developed the following model that outlines how cDNA synthesis occurs in the absence and presence of RNA using standard TS oligo vs. iso3TS oligo (Figure 1)
Summary
The template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. For investigators interested in transcriptome analysis and gene discovery from non-model organisms or those interested in transcriptional processes at a cellspecific level, TS-PCR cDNA synthesis represents the most straightforward approach to generating cDNA for sequencing efforts. This approach does suffer from some drawbacks, including the potential for high background that interferes with downstream sequencing efficiencies when starting with very small quantities of RNA
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