Giraldez, Maria DSpengler, Ryan MEtheridge, AltonGoicochea, Annika JTuck, MissyChoi, Sung WonGalas, David JTewari, Muneesh2023-01-252023-01-252019-05-03http://hdl.handle.net/10668/13912Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non-coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are 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-RNA-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-RNA-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 hematopoietic stem cell transplant patients, bone marrow- and liver-enriched exRNA genes were 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-RNA-seq opens up new possibilities for plasma transcriptomic biomarker development.enAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/RNA‐seqcell‐free RNAextracellular RNAliquid biopsyBiomarkersBlood Chemical AnalysisCell-Free Nucleic AcidsComputational BiologyGene Expression ProfilingHumansMicroRNAsRNA, Long NoncodingRNA, MessengerRNA-SeqSequence Analysis, RNAresearch article31053596open access10.15252/embj.20191016951460-2075PMC6545557https://doi.org/10.15252/embj.2019101695https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545557/pdf