ArticleBMC genomics2010
Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples.
Article in BMC genomics, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 40 papers.
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40 citing papers in PubMed.
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- Imprinted X chromosome inactivation at the gamete-to-embryo transition.Molecular cell · 2024Article
- Clamping-mediated incorporation of single-stranded DNA with concomitant DNA synthesis by Taq polymerase involves nick-translation.Scientific reports · 2024Article
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- Sequencing of Transcriptome-Wide Poly(A) Tails by PAIso-seq.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Transcriptome-wide measurement of poly(A) tail length and composition at subnanogram total RNA sensitivity by PAIso-seq.Nature protocols · 2022Review
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- A Molecular Analysis of Memory B Cell and Antibody Responses AgainstFrontiers in immunology · 2022Article
- Global approaches for profiling transcription initiation.Cell reports methods · 2021Review
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4 authors.
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No grant is acknowledged in the PubMed record.
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.
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