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

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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 extracellular RNAs (exRNAs) isolated from serum and platelet-poor EDTA, acid citrate dextrose (ACD), and heparin plasma to study the effect on ex-mRNA capture. Compared with conventional sRNA-seq, PNK treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by RNA originating from 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 350–1100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2300. EDTA and ACD plasma showed a destabilizing effect on ex‑mRNA and noncoding RNA ribonucleoprotein complexes compared with other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome 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.

Authors

Kemal M. Akat, Youngmin A. Lee, Arlene Hurley, Pavel Morozov, Klaas E.A. Max, Miguel Brown, Kimberly Bogardus, Anuoluwapo Sopeyin, Kai Hildner, Thomas G. Diacovo, Markus F. Neurath, Martin Borggrefe, Thomas Tuschl