Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

• Published in: JCI insight Vol. 5; no. 9
• Main Authors: Akat, Kemal M, Lee, Youngmin A, Hurley, Arlene, Morozov, Pavel, Max, Klaas Ea, Brown, Miguel, Bogardus, Kimberly, Sopeyin, Anuoluwapo, Hildner, Kai, Diacovo, Thomas G, Neurath, Markus F, Borggrefe, Martin, Tuschl, Thomas
• Format: Journal Article
• Language: English
• Published: United States American Society for Clinical Investigation 02.05.2019
• Subjects: Technical Advance; Molecular diagnosis; Cardiology; RNA processing; Bioinformatics; Vascular Biology
• ISSN: 2379-3708; 2379-3708
• DOI: 10.1172/jci.insight.127317

Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt reads originated from the coding sequences, in a pattern reminiscent of ribosome-profiling. Blood sample type had a considerable influence on the exRNA profile. On average approximately 350 to 1,100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2,300. EDTA and ACD plasma showed a destabilizing effect on ex mRNA and non-coding RNA ribonucleoprotein complexes compared to other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome (ACS) and healthy controls. The improved tissue resolution of ex mRNAs after PNK-treatment enabled us to detect a neutrophil-signature in ACS that escaped detection by ex miRNA analysis.