Phospho-sRNA-seq reveals extracellular mRNA/lncRNA fragments as potential biomarkers in human plasma

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Whereas extracellular microRNAs (miRNAs) in blood plasma are extensively characterized, extracellular messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are less well-studied. We report that plasma contains fragmented mRNAs and lncRNAs that are largely missed by standard small RNA-seq protocols due to lack of 5’ phosphate or presence of 3’ phosphate. These fragments were revealed using a modified protocol (“phospho-sRNA-seq”) incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5’ and 3’ ends, as well as human plasma exRNA. Analyzing phospho-sRNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of bone marrow transplant patients, bone marrow-and liver-enriched exRNA genes tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-sRNA-seq opens up new possibilities for plasma transcriptomic biomarker development.