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

JCI Insight. 2019 Apr 11;5(9):e127317. doi: 10.1172/jci.insight.127317.

Abstract

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.

Keywords: Bioinformatics; Cardiology; Molecular diagnosis; RNA processing; Vascular Biology.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acute Coronary Syndrome / blood
  • Acute Coronary Syndrome / genetics*
  • Adult
  • Aged
  • Biomarkers / blood
  • Blood Cells / metabolism*
  • Case-Control Studies
  • Cell-Free Nucleic Acids / blood
  • Cell-Free Nucleic Acids / genetics*
  • Citric Acid
  • Edetic Acid
  • Erythrocytes / metabolism
  • Female
  • Glucose / analogs & derivatives
  • Heparin
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Leukocytes, Mononuclear / metabolism
  • Liver / metabolism*
  • Male
  • Middle Aged
  • Monocytes / metabolism
  • Muscle, Skeletal / metabolism*
  • Neutrophils / metabolism
  • Plasma
  • Polynucleotide 5'-Hydroxyl-Kinase
  • Proof of Concept Study
  • RNA-Seq / methods*
  • Sequence Analysis, RNA / methods
  • Serum
  • Specimen Handling

Substances

  • Biomarkers
  • Cell-Free Nucleic Acids
  • acid citrate dextrose
  • Citric Acid
  • Heparin
  • Edetic Acid
  • Polynucleotide 5'-Hydroxyl-Kinase
  • Glucose