Diurnal stability of cell-free DNA and cell-free RNA in human plasma samples.
Author(s): Wagner JT, Kim HJ, Johnson-Camacho KC, Kelley T, Newell LF, Spellman PT, Ngo TTM
Publication: Sci Rep, 2020, Vol. 10, Page 16456
PubMed ID: 33020547 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of time of blood collection and compared analytical methods on the measured levels of cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in plasma.
Conclusion of Paper
No significant differences in cfDNA or cfRNA levels were found over the course of the day or between the two days, regardless of analytical method. cfDNA levels differed significantly among individuals but no significant differences in mRNA levels were observed between individuals. Measured cfDNA concentrations were very strongly correlated between the analytical methods.
Studies
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Study Purpose
The purpose of this study was to investigate the effects of time of blood collection and analytical method on the measured levels of cfDNA and cfRNA in plasma. EDTA blood was collected intravenously (IV) or by venipuncture (when IV failed) from four healthy donors (three females, one male) five times at 2 h 45 min intervals over the course of a day and then again one week later. Plasma was obtained by room temperature centrifugation at 1000 × g for 10 min followed by 25000 x g for 10 min and stored as 1 mL aliquots at -80°C. cfDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit and cfRNA was extracted using the Plasma/Serum Circulating and Exosomal RNA Purification Kit. Extracted cfRNA was treated with MBU of Baseline-ZERO DNase in 1X Baseline-ZERO and purified using the Zymo RNA Clean & Concentrator-5 Kit. cfDNA was quantified using the Qubit dsDNA HS Assay Kit on a fluorometer and by duplex ddPCR amplification of TERT and NAGK. Total cfRNA was quantified using the Agilent RNA 6000 Pico Kit and after reverse transcription by ddPCR of ACTB and GAPDH.
Summary of Findings:
While fluctuations in cfDNA levels were observed, no significant differences in cfDNA concentrations were found over the course of the day or between the days of collection as quantified by fluorometer or ddPCR. Similarly, total cfRNA and levels of GAPDH and ACTB were comparable among the timepoints and days. cfDNA levels as quantified by fluorometer and ddPCR differed significantly among individuals (P<0.05), and while variation in GAPDH mRNA levels attributable to individual were observed, none of the individual comparisons reached significance. Measured cfDNA concentrations were very strongly correlated between the analytical methods (ρ=0.97, P<0.0001).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry RNA Digital PCR RNA Automated electrophoresis/Bioanalyzer DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Day 1
Day 8
8:30 AM
11:15 AM
2:00 PM
4:45 PM
7:30 PM
Digital PCR Specific Targeted nucleic acid TERT
NAGK
GAPDH
ACTB
Digital PCR Specific Technology platform Bioanalyzer
Fluorometer