Phospho-RNA-seq: a modified small RNA-seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma.

Maria D Giraldez,David J Galas,Missy Tuck,Ryan M Spengler,Annika J Goicochea,Muneesh Tewari,Sung Won Choi,Alton Etheridge

doi:10.15252/embj.2019101695

Abstract

Extracellular 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.

Highlights

In recent years, the discovery of a variety of extracellular RNA molecules present in the human bloodstream and other biofluids has been of great interest given their potential value as minimally-invasive biomarkers for a wide range of diseases (Freedman et al, 2016; Max et al, 2018; Godoy et al, 2018; Yuan et al, 2016)
We report that plasma contains fragmented messenger RNAs (mRNA) and long non-coding RNAs (lncRNA) that are missed by standard small RNAseq protocols due to lack of 5’ phosphate or presence of 3’ phosphate
After having demonstrated that phospho-RNA-seq combined with a stringent pipeline is critical for recovering mRNA and lncRNA fragments in plasma, we examined the efficiency of this strategy for capturing microRNAs from plasma by analyzing mature microRNA read counts from healthy donor plasma samples prepared with and without PNK treatment

Summary

Introduction

The discovery of a variety of extracellular RNA (exRNA) molecules present in the human bloodstream and other biofluids has been of great interest given their potential value as minimally-invasive biomarkers for a wide range of diseases (Freedman et al, 2016; Max et al, 2018; Godoy et al, 2018; Yuan et al, 2016). RNA-seq has transformed transcriptome characterization in a wide range of biological contexts (Mortazavi et al, 2008; Wang et al, 2009) including its application to analyze exRNA in body fluids (Adiconis et al, 2013; Giraldez et al, 2018) These efforts have begun to elucidate the complex composition of exRNA in blood (Freedman et al, 2016; Max et al, 2018; Yeri et al, 2017; Godoy et al, 2018). These profiling studies have used a variety of methods to evaluate exRNA (e.g. microarrays and different methodologies for RNAseq) which, not surprisingly, contributes to the variation in findings across the studies

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