In contrast to general belief, a substantial part of the human protein coding transcriptome is abundantly present in the blood as extracellular mRNA, ready to exploited. It is well known that cancer cells actively and passively release RNA cargo into circulation and their detection may inform on the patient disease status.
We developed and applied a probe based RNA capture sequencing method as a sensitive RNA sequencing workflow to study thousands of transcripts in cell-free RNA from cancer patients’ plasma. The method is based on exome-style enrichment of a randomly primed cDNA library with preservation of strandedness information. More than one million capture probes target 21,000 human messenger RNA and 60,000 human long non-coding RNA genes. Apart from RNA abundance profiling, this type of data can also be used to detect structural RNA variants, such as somatic mutations, fusion genes, and RNA editing events, all known to play an important role in cancer.
On average, between 6000-10,000 RNA genes are reproducibly detected in 0.2 ml of plasma. Detection and coverage sensitivity is greatly increased by using larger plasma volume and improved adaptor ligation strategies. We also observed a positive correlation between number of platelets in plasma and detected genes and variants, in line with their tumor-educated nature. Our benchmarked RNA variant pipeline identifies thousands of germline and somatic variants in circulating mRNA. A dedicated titration experiment in which plasma from cancer and healthy individuals were mixed in known ratios demonstrates excellent quantitative performance. Pronounced RNA abundance differences and enriched pathways are observed between cancer types and during treatment. The RNA capture sequencing also works on other body fluids, such as urine and serum, and simultaneous targeting of mRNA and lncRNA provides substantial enrichment of otherwise low-abundant lncRNAs.
RNA capture sequencing of liquid biopsies is a promising new application to support precision oncology and is expected to enhance therapy stratification, treatment response monitoring and early detection of relapse.
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Biogazelle and Ghent University
J. Vandesompele: Apart from professorship at Ghent University, co-founder and part-time CSO at Biogazelle, a Ghent University spin-off company.