Quantitative Transcriptomics using Designed Primer-based Amplification

Quantitative Transcriptomics using Designed Primer-based Amplification

We developed a novel Designed Primer-based RNA-sequencing strategy (DP-seq) that uses a defined set of heptamer primers to amplify the majority of expressed transcripts from limiting amounts of mRNA, while preserving their relative abundance. Our strategy reproducibly yielded high levels of amplific...

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Main Authors: Bhargava, Vipul, Ko, Pang, Willems, Erik, Mercola, Mark, Subramaniam, Shankar
Format: Online
Language:English
Published: Nature Publishing Group 2013
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638165/
id pubmed-3638165
recordtype oai_dc
spelling pubmed-36381652013-04-29 Quantitative Transcriptomics using Designed Primer-based Amplification Bhargava, Vipul Ko, Pang Willems, Erik Mercola, Mark Subramaniam, Shankar Article We developed a novel Designed Primer-based RNA-sequencing strategy (DP-seq) that uses a defined set of heptamer primers to amplify the majority of expressed transcripts from limiting amounts of mRNA, while preserving their relative abundance. Our strategy reproducibly yielded high levels of amplification from as low as 50 picograms of mRNA while offering a dynamic range of over five orders of magnitude in RNA concentrations. We also demonstrated the potential of DP-seq to selectively suppress the amplification of the highly expressing ribosomal transcripts by more than 70% in our sequencing library. Using lineage segregation in embryonic stem cell cultures as a model of early mammalian embryogenesis, DP-seq revealed novel sets of low abundant transcripts, some corresponding to the identity of cellular progeny before they arise, reflecting the specification of cell fate prior to actual germ layer segregation. Nature Publishing Group 2013-04-29 /pmc/articles/PMC3638165/ /pubmed/23624976 http://dx.doi.org/10.1038/srep01740 Text en Copyright © 2013, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by-nc-nd/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Bhargava, Vipul
Ko, Pang
Willems, Erik
Mercola, Mark
Subramaniam, Shankar
spellingShingle Bhargava, Vipul
Ko, Pang
Willems, Erik
Mercola, Mark
Subramaniam, Shankar
Quantitative Transcriptomics using Designed Primer-based Amplification
author_facet Bhargava, Vipul
Ko, Pang
Willems, Erik
Mercola, Mark
Subramaniam, Shankar
author_sort Bhargava, Vipul
title Quantitative Transcriptomics using Designed Primer-based Amplification
title_short Quantitative Transcriptomics using Designed Primer-based Amplification
title_full Quantitative Transcriptomics using Designed Primer-based Amplification
title_fullStr Quantitative Transcriptomics using Designed Primer-based Amplification
title_full_unstemmed Quantitative Transcriptomics using Designed Primer-based Amplification
title_sort quantitative transcriptomics using designed primer-based amplification
description We developed a novel Designed Primer-based RNA-sequencing strategy (DP-seq) that uses a defined set of heptamer primers to amplify the majority of expressed transcripts from limiting amounts of mRNA, while preserving their relative abundance. Our strategy reproducibly yielded high levels of amplification from as low as 50 picograms of mRNA while offering a dynamic range of over five orders of magnitude in RNA concentrations. We also demonstrated the potential of DP-seq to selectively suppress the amplification of the highly expressing ribosomal transcripts by more than 70% in our sequencing library. Using lineage segregation in embryonic stem cell cultures as a model of early mammalian embryogenesis, DP-seq revealed novel sets of low abundant transcripts, some corresponding to the identity of cellular progeny before they arise, reflecting the specification of cell fate prior to actual germ layer segregation.
publisher Nature Publishing Group
publishDate 2013
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638165/
_version_ 1611973300799406080

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