Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples
Open accessAbstract
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.
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ERCC spike-ins are not capped at the 5' end, which may reduce template-switching efficiency compared with endogenous mRNAs.
“Furthermore, the ERCC spike-ins are not capped at the 5’ end, which may further lead to reduced template switching efficiency (used in several protocols) as compared to endogenous mRNAs14.”
From Power analysis of single-cell RNA-sequencing experiments (2017) · cites this paper as [14]
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- Authors
- Kapteyn, J., He, R., McDowell, E. T., & Gang, D. R.
- Affiliations
- University of Arizona (US)
- Corresponding author
- Gang, David R
- Venue
- BMC Genomics
- Published
- 2010-07-02
- Volume
- 11
- Issue
- 1
- Pages
- 413-413
- ISSN
- 1471-2164 (ISSN-L)
- PMC
- PMC2996941
- OpenAlex
- W2165392372
- Cited by
- 59
- References
- 22
- Citations by year
- 2026: 5 · 2025: 6 · 2024: 4 · 2023: 2 · 2022: 5 · 2021: 2 · 2020: 8 · 2019: 8 · 2018: 1 · 2017: 3 · 2016: 3 · 2014: 3 · 2013: 1 · 2012: 4
- FWCI
- 1.17
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- BMC Genomics (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- National Science Foundation (0604336, 0820346)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- cDNA synthesis, background suppression, single-cell RNA sequencing, 454 pyrosequencing, laser microdissection
- MeSH terms
- Base Sequence, Isomerism, Molecular Sequence Data, Moloney murine leukemia virus, Oligonucleotides, Plants, RNA-Directed DNA Polymerase, RNA, Gene Library, Polymerase Chain Reaction, Sequence Analysis, DNA, DNA, Complementary, Plant Cells
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