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Whole transcriptome profiling reveals the RNA content of motor axons

Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well establi...

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Autores principales: Briese, Michael, Saal, Lena, Appenzeller, Silke, Moradi, Mehri, Baluapuri, Apoorva, Sendtner, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770199/
https://www.ncbi.nlm.nih.gov/pubmed/26464439
http://dx.doi.org/10.1093/nar/gkv1027
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author Briese, Michael
Saal, Lena
Appenzeller, Silke
Moradi, Mehri
Baluapuri, Apoorva
Sendtner, Michael
author_facet Briese, Michael
Saal, Lena
Appenzeller, Silke
Moradi, Mehri
Baluapuri, Apoorva
Sendtner, Michael
author_sort Briese, Michael
collection PubMed
description Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.
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spelling pubmed-47701992016-02-29 Whole transcriptome profiling reveals the RNA content of motor axons Briese, Michael Saal, Lena Appenzeller, Silke Moradi, Mehri Baluapuri, Apoorva Sendtner, Michael Nucleic Acids Res Methods Online Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts. Oxford University Press 2016-02-29 2015-10-12 /pmc/articles/PMC4770199/ /pubmed/26464439 http://dx.doi.org/10.1093/nar/gkv1027 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Briese, Michael
Saal, Lena
Appenzeller, Silke
Moradi, Mehri
Baluapuri, Apoorva
Sendtner, Michael
Whole transcriptome profiling reveals the RNA content of motor axons
title Whole transcriptome profiling reveals the RNA content of motor axons
title_full Whole transcriptome profiling reveals the RNA content of motor axons
title_fullStr Whole transcriptome profiling reveals the RNA content of motor axons
title_full_unstemmed Whole transcriptome profiling reveals the RNA content of motor axons
title_short Whole transcriptome profiling reveals the RNA content of motor axons
title_sort whole transcriptome profiling reveals the rna content of motor axons
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770199/
https://www.ncbi.nlm.nih.gov/pubmed/26464439
http://dx.doi.org/10.1093/nar/gkv1027
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