Profiling extracellular long RNA transcriptome in human plasma and extracellular vesicles for biomarker discovery

The recent discovery of extracellular RNAs in blood, including RNAs in extracellular vesicles (EVs), combined with low-input RNA-sequencing advances have enabled scientists to investigate their role in human disease. To date, most studies have been focusing on small RNAs, and methodologies to optimize long RNAs measurement are lacking. We used plasma RNA to assess the performance of six long RNA sequencing methods, at two different sites, and we report their differences in reads (%) mapped to the genome/transcriptome, number of genes detected, long RNA transcript diversity, and reproducibility. Using the best performing method, we further compare the profile of long RNAs in the EV- and no-EV-enriched RNA plasma compartments. These results provide insights on the performance and reproducibility of commercially available kits in assessing the landscape of long RNAs in human plasma and different extracellular RNA carriers that may be exploited for biomarker discovery.近年来，血液中细胞外RNA（extracellular RNAs）的相关发现——包括存在于细胞外囊泡（extracellular vesicles, EVs）中的RNA——结合低起始量RNA测序（low-input RNA-sequencing）技术的长足进步，使得科学家得以探究其在人类疾病中的作用。迄今为止，绝大多数相关研究均聚焦于小RNA领域，而用于优化长RNA检测的成熟方法仍较为匮乏。我们采用血浆RNA样本，在两个不同实验站点对六种长RNA测序方法的性能进行了评估，并报告了各方法在比对至基因组/转录组的读段占比、检测到的基因数量、长RNA转录本多样性以及实验可重复性方面的差异。借助性能最优的测序方法，我们进一步对比了富集细胞外囊泡（EVs）与非富集细胞外囊泡的血浆RNA组分中的长RNA表达谱。本研究结果可为商用试剂盒在评估人类血浆及不同细胞外RNA载体中的长RNA表达图谱方面的性能与可重复性提供参考，相关发现可用于生物标志物（biomarker）的挖掘研究。