Abstract
RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.
Highlights
All human biofluids contain a multitude of extracellular nucleic acids, harboring a wealth of information about health and disease status
As we will illustrate below, ePRP has a higher RNA input concentration, which explains the lower number of duplicate reads compared to ePFP
EPRP contains approximately twice as many uniquely mapped reads, possibly the result of more intact RNA in platelets. When only considering these unique reads, more than 75% of them derived from mitochondrial RNA in ePRP (Fig. 2B)
Summary
All human biofluids contain a multitude of extracellular nucleic acids, harboring a wealth of information about health and disease status. Only a few whole transcriptome profiling attempts were made on urine, plasma or extracellular vesicles[5,6,7,8,9], quantifying both polyadenylated and non-polyadenylated RNA transcripts (Table 1). All these methods suffer from one or more limitations such as short fragment length, low number of quantified genes or a high level of ribosomal RNA contamination. We aimed to assess the performance of a strand-specific total RNA library preparation method for different types of biofluids and derived extracellular vesicles (EVs). Low-input volumes are technically feasible and the method allows the detection of several thousand genes of different classes
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