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Targeted transcript profiling by sequencing

In this work we present a targeted gene expression strategy employing trinucleotide threading (TnT) amplification and massive parallel sequencing. We have previously shown that TnT combined with array readout accurately monitors expression levels. However, with this detection strategy spurious produ...

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Detalles Bibliográficos
Autores principales: Zajac, Pawel, Ahmadian, Afshin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492873/
https://www.ncbi.nlm.nih.gov/pubmed/23139866
http://dx.doi.org/10.1038/srep00821
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author Zajac, Pawel
Ahmadian, Afshin
author_facet Zajac, Pawel
Ahmadian, Afshin
author_sort Zajac, Pawel
collection PubMed
description In this work we present a targeted gene expression strategy employing trinucleotide threading (TnT) amplification and massive parallel sequencing. We have previously shown that TnT combined with array readout accurately monitors expression levels. However, with this detection strategy spurious products go undetected. Accordingly, we adapted the TnT protocol to massive parallel sequencing to acquire an unbiased view of the entire TnT-generated product population. In this manner we investigated the identity of undesired products, their extent at different oligonucleotide:RNA ratios and their effect on the expression levels. We demonstrate that TnT gene expression profiling with massive sequencing readout renders reliable expression data from as low as 3.5 ng of total RNA. Moreover, using 350 ng of total RNA results in only 0.7% to 1.1% undesired products. When lowering the amount of input material, the undesired product fraction increases but this does not influence the expression profiles.
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spelling pubmed-34928732012-11-08 Targeted transcript profiling by sequencing Zajac, Pawel Ahmadian, Afshin Sci Rep Article In this work we present a targeted gene expression strategy employing trinucleotide threading (TnT) amplification and massive parallel sequencing. We have previously shown that TnT combined with array readout accurately monitors expression levels. However, with this detection strategy spurious products go undetected. Accordingly, we adapted the TnT protocol to massive parallel sequencing to acquire an unbiased view of the entire TnT-generated product population. In this manner we investigated the identity of undesired products, their extent at different oligonucleotide:RNA ratios and their effect on the expression levels. We demonstrate that TnT gene expression profiling with massive sequencing readout renders reliable expression data from as low as 3.5 ng of total RNA. Moreover, using 350 ng of total RNA results in only 0.7% to 1.1% undesired products. When lowering the amount of input material, the undesired product fraction increases but this does not influence the expression profiles. Nature Publishing Group 2012-11-08 /pmc/articles/PMC3492873/ /pubmed/23139866 http://dx.doi.org/10.1038/srep00821 Text en Copyright © 2012, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by-nc-sa/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
spellingShingle Article
Zajac, Pawel
Ahmadian, Afshin
Targeted transcript profiling by sequencing
title Targeted transcript profiling by sequencing
title_full Targeted transcript profiling by sequencing
title_fullStr Targeted transcript profiling by sequencing
title_full_unstemmed Targeted transcript profiling by sequencing
title_short Targeted transcript profiling by sequencing
title_sort targeted transcript profiling by sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492873/
https://www.ncbi.nlm.nih.gov/pubmed/23139866
http://dx.doi.org/10.1038/srep00821
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