Abstract

Many emerging technologies are reliant on circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) applications in the clinic. However, the impact of diurnal cycles or daily meals on circulating analytes are poorly understood and may be confounding factors when developing diagnostic platforms. To begin addressing this knowledge gap, we obtained plasma from four healthy donors serially sampled five times during 12 h in a single day. For all samples, we measured concentrations of cfDNA and cfRNA using both bulk measurements and gene-specific digital droplet PCR. We found no significant variation attributed to blood draw number for the cfDNA or cfRNA. This indicated that natural diurnal cycles and meal consumption do not appear to significantly affect abundance of total cfDNA, total cfRNA, or our two selected cfRNA transcripts. Conversely, we observed significant variation between individual donors for cfDNA and one of the cfRNA transcripts. The results of this work suggest that it will be important to consider patient-specific baselines when designing reliable circulating cfDNA or cfRNA clinical assays.

Highlights

  • Liquid-biopsy based diagnostic platforms are highly desired and increasingly accepted in clinical ­settings[1]

  • Using this digital droplet PCR (ddPCR) approach, we targeted two single-copy genomic DNA regions as proxies for cell-free DNA (cfDNA) abundance: one locus containing the gene telomerase reverse transcriptase (TERT) and one locus containing the gene N-acetylglucosamine kinase (NAGK); for cell-free RNA (cfRNA) we targeted two commonly used genes used for mRNA normalization: β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

  • Confounding factors related to meals, time of day, and the intrinsic interpersonal variation could critically affect analyte abundance, normalization, and multi-omic integration. To begin addressing these concerns, here we provide the first descriptions of diurnal cfDNA and cfRNA derived from the same cohort

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Summary

Introduction

Liquid-biopsy based diagnostic platforms are highly desired and increasingly accepted in clinical ­settings[1]. The sensitivity of current methods to reliably characterize nucleotide sequences and subcellular particles at single-event resolution has generated substantial interest in utilizing circulating cell-free DNA (cfDNA, e.g.7–10) and cell-free RNA (cfRNA, e.g.11–15) as clinically-relevant biomarkers. Human plasma contains both vesicular and extravesicular RNA and DNA; these different components may have distinctive contents with potential clinical ­relevance[16]. Our results suggest that while cfDNA and cfRNA are overall stably expressed diurnally, several of the analytes demonstrate significant interpersonal or daily variation Deeper characterization of these sources of variation will likely be required before the circulating analytes gain greater acceptance as clinically practical liquid-biopsy platforms

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