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Nucleic Acids Res. 2016 Feb 29;44(4):e33. doi: 10.1093/nar/gkv1027. Epub 2015 Oct 12.

Whole transcriptome profiling reveals the RNA content of motor axons.

Author information

1
Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
2
Core Unit Systems Medicine, University of Wuerzburg, 97080 Wuerzburg, Germany.
3
Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany Sendtner_M@ukw.de.

Abstract

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.

PMID:
26464439
PMCID:
PMC4770199
DOI:
10.1093/nar/gkv1027
[Indexed for MEDLINE]
Free PMC Article

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