Work

Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples

Year: 2010

Type: article

Abstract: BACKGROUND: 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. 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. RESULTS: In 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. CONCLUSIONS: Our 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.

Source: BMC Genomics

DOI: 10.1186/1471-2164-11-413

Authors: Jeremy Kapteyn, Ruifeng He, Eric T McDowell, David R Gang

Institutions: University of Arizona, BIO5 Institute, Washington State University

Language: English

Topic: Molecular Biology Techniques and Applications

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,090

Funder: National Science Foundation