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

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Extracellular RNAs (exRNAs) are emerging as critical biomarkers.
• Extracellular messenger RNAs (ex-mRNAs) may offer advantages over extracellular microRNAs (ex-miRNAs).
• Optimizing exRNA isolation and analysis is crucial for biomarker discovery.

Purpose of the Study:

• To evaluate the impact of T4 polynucleotide kinase (PNK) end-treatment on extracellular messenger RNA (ex-mRNA) capture using small-RNA sequencing (sRNA-seq).
• To investigate the influence of different blood sample types (serum, EDTA, ACD, heparin plasma) on exRNA profiles.
• To assess the utility of PNK-treated ex-mRNAs for detecting disease-specific signatures, exemplified by acute coronary syndrome (ACS).

Main Methods:

• Isolation of total exRNA from serum and platelet-poor plasma (EDTA, ACD, heparin).
• Comparison of conventional sRNA-seq with sRNA-seq employing T4 PNK end-treatment.
• Analysis of exRNA profiles, including read length distribution, origin (coding vs. non-coding), and cell-type specific contributions.
• Proof-of-concept study comparing exRNA profiles of ACS patients and healthy controls.

Main Results:

• PNK treatment increased ex-mRNA read detection by up to 50-fold compared to conventional sRNA-seq.
• The exRNA pool was primarily derived from hematopoietic cells, platelets, and the liver.
• Blood sample type significantly influenced exRNA profiles, with serum yielding a higher number of distinct ex-mRNA transcripts.
• EDTA and ACD plasma exhibited a destabilizing effect on exRNA ribonucleoprotein complexes.
• PNK-treated ex-mRNAs revealed a neutrophil signature in ACS patients that was not detected by ex-miRNA analysis.

Conclusions:

• T4 PNK end-treatment substantially enhances the capture and detection of informative ex-mRNAs.
• Blood sample type critically impacts exRNA profiling, necessitating careful consideration for standardization.
• PNK-enhanced ex-mRNA sequencing provides improved tissue resolution and holds significant potential for identifying novel disease biomarkers, such as the neutrophil signature in ACS.