title: Whole transcriptome profiling reveals the RNA content of motor axons.
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.
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Briese
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Saal
authors.affiliation: Core Unit Systems Medicine, University of Wuerzburg, 97080 Wuerzburg, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Appenzeller
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Mehri
authors.initials: M
authors.lastname: Moradi
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Apoorva
authors.initials: A
authors.lastname: Baluapuri
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany Sendtner_M@ukw.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Sendtner
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D009046:Motor Neurons / Q000378:metabolism; D061251:Primary Cell Culture; D062085:RNA, Long Noncoding / Q000096:biosynthesis / Q000235:genetics*; D012335:RNA, Ribosomal / Q000096:biosynthesis / Q000235:genetics*; D020733:RNA, Small Cytoplasmic / Q000096:biosynthesis / Q000235:genetics; D017423:Sequence Analysis, RNA; D018271:Signal Recognition Particle / Q000096:biosynthesis / Q000235:genetics; D059467:Transcriptome / Q000235:genetics*
chemical_list: C115990:7SL RNA; D062085:RNA, Long Noncoding; D012335:RNA, Ribosomal; D020733:RNA, Small Cytoplasmic; D018271:Signal Recognition Particle