Phospho-RNA-seq for plasma transcriptome profiling

Maria Giraldez ^1,2,3 Ryan Spengler ¹ Alton Etheridge ⁴ Annika Goicochea ¹ Missy Tuck ¹ Sung Choi ⁵ David Galas ⁴ Muneesh Tewari ^1,6,7

¹Department of Internal Medicine, Hematology/Oncology Division, University of Michigan, Ann Arbor, Michigan, USA
²Institute of Biomedicine of Seville (IBiS), Institute of Biomedicine of Seville (IBiS), Seville, Spain
³Unit of Digestive Diseases, Virgen del Rocio University Hospital, Seville, Spain
⁴Pacific Northwest Research Institute, Pacific Northwest Research Institute, Seattle, Washington, USA
⁵Department of Pediatrics, Hematology/Oncology Division, University of Michigan, Ann Arbor, Michigan, USA
⁶Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA
⁷Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA

Cell-free RNA in plasma has been an active area of research, with most studies to date focusing on cell-free microRNAs. Studies of cell-free messenger RNA (mRNA) and long non‐coding RNA (lncRNA) have been more limited, with some controversy about the nature of such circulating transcripts in plasma. We present a modified RNA-seq methodology, called phospho-RNA-seq, which, when combined with a stringent computational pipeline, revealed thousands of mRNA and lncRNAs in blood plasma. The key to this approach is the incorporation of T4-polynucleotide kinase treatment, which enabled the recovery of fragmented mRNA and lncRNA transcripts that are otherwise missed by standard small RNA-seq protocols because they lack a 5`-phosphate and/or have a 3`-phosphate. Phospho-RNA-seq enabled the identification of sets of mRNA/lncRNA transcripts in plasma, including gene sets expressed in a tissue-specific manner. As proof-of-concept validation of the approach for biomarker identification, we used phospho-RNA-seq to longitudinally profile plasma specimens collected from patients undergoing hematopoietic stem cell transplantation. We detected bone marrow-enriched and liver-enriched transcript sets in plasma that these tracked with bone marrow recovery and hepatic injury, respectively. By providing expanded access to the plasma transcriptome, phospho-RNA-seq enables the discovery of new cell-free RNA biomarker signatures for a wide variety of clinical applications.