Briese Michael, Saal Lena, Appenzeller Silke, Moradi Mehri, Baluapuri Apoorva, Sendtner Michael
Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
Core Unit Systems Medicine, University of Wuerzburg, 97080 Wuerzburg, Germany.
Nucleic Acids Res. 2016 Feb 29;44(4):e33. doi: 10.1093/nar/gkv1027. Epub 2015 Oct 12.
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.
大多数存在于极化细胞（如神经元）内的RNA会以一种协调的方式在亚细胞水平上进行分选。尽管在从少量输入RNA中分析多聚腺苷酸化RNA的方法开发方面取得了进展，但同时分析编码RNA和非编码RNA的技术尚未完善。在此，我们优化了一种基于双随机引物的转录组分析方法，并将其应用于低至10 pg的系列稀释总RNA。重复样本之间表达基因的读数计数具有很强的相关性，表明该方法具有可重复性和可扩展性。我们的转录组分析方法检测到了大小>300个碱基的编码RNA和长链非编码RNA。与使用传统方法的总RNA测序相比，我们的方案由于减少了核糖体RNA的捕获，检测到的基因多了70%。我们使用该方法分析了分隔的运动神经元的RNA组成。树突状细胞区域富含具有突触后功能的转录本以及某些核非编码RNA，如7SK。在轴突中，与翻译相关的转录本富集，包括细胞质非编码RNA 7SL。我们的分析方法可应用于广泛的研究，包括神经退行性疾病中亚细胞转录组的扰动以及对显微切割组织样本（如解剖学上定义的纤维束）的研究。
