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Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization.

Author(s): Sandberg F, Kueng N, Largiadèr CR, Amstutz U

Publication: Sci Rep, 2025, Vol. 15, Page 22999

PubMed ID: 40596083 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared two different methods to extract cell-free DNA from urine and investigated the variability associated with the individual, sex, the researcher performing the extraction, the extraction itself and the assay. Additionally, the effect of urine volume on cfDNA yield and the number of copies of AGO1 were investigated. Urine was collected from three healthy men and three healthy women into LoBind tubes containing Streck Urine Preserve on two different days. Urine was centrifuged at 2000 g for 15 min and the supernatant was stored at -80°C. Creatinine was quantified in each urine specimen. Some urine from the three men and three women were pooled (a pool of urine from males and a separate pool of urine from females), aliquoted, spiked with 85,000 copies of a 180 bp fragment of CERBIS, and cfDNA extraction was performed in triplicate on three separate days by three different researchers and then assayed in triplicate. To compare intra- versus inter-individual variability, the remaining individual specimens were spiked with 45,000 copies each of a 180 bp and 89 bp fragment of CERBIS, and the cfDNA was extracted in triplicate using both kits and then assayed in triplicate. cfDNA was extracted from urine using the Zymo Quick-DNA Urine Kit or an in-house method using Q Sepharose Fast Flow and Micro Bio-Spin Chromatography Columns, and clean-up was with QIAquick Spin Columns. cfDNA was quantified using the Qubit 1X dsDNA HS Assay Kit and by droplet digital PCR (ddPCR) amplification of AGO1. The size distribution of the isolated cfDNA was analyzed using the High Sensitivity DNA Kit with the 2100 Bioanalyzer system.

   Summary of Findings:

   Following extraction using Q-sepharose, the cfDNA yield and number of AGO1 copies were correlated with the urine input volume (r=0.995, p<0.001 and r=0.999, p<0.001, respectively). The size distribution pattern of the urinary cfDNA was not correlated with the urine input volume as itdiffered between Q-sepharose and the Zymo Quick-DNA Urine Kit (Zymo) extraction methods. Following extraction with Q-sepharose, there were more fragments < 90 bp, less high molecular weight DNA (> 1,000 bp), and more fragments that were 89 bp in length relative to those that were 180 bp than when extraction was with the Zymo Kit. The Zymo Kit also failed to isolate the 50 bp DNA from phosphate buffered saline (PBS) spiked with a DNA ladder. However, mean cfDNA concentration was higher when extracted with the Zymo Kit relative to the Q-sepharose method (3.72 versus 2.85 ng/mL urine). When potential sources of variability were investigated using a spiked-in 180 bp fragment of CEREBIS that was extracted and assayed in triplicate from the urine of three men and three women, the largest variability was between individuals (96.17% with Q-sepharose extraction and 89.03% with Zymo extraction), followed by between males and females (3.59% for Q-sepharose extractions and 10.92% for Zymo extractions), with only negligible amounts of variability associated with inter- or intra- extraction (<0.5% all). Similarly, when the urine was spiked with both 89 and 180 bp fragments of CEREBIS, the majority of the variability occurred between individuals (95.51% with Q-sepharose extraction and 86.23% with Zymo extraction), but while variability between males and females continued to have a larger effect than intra- extraction variability when extracted with the Zymo Kit (14.49% versus 0.27%), inter-extraction variability (not assessed with Zymo Kut) was higher than the variability that occurred between specimens from men and women when extracted with Q-sepharose (5.25% versus 0.00%). Importantly, the mean extraction efficiency for isolation of the 180 bp and 89 bp spiked-in DNA were higher using the Zymo (61.12% and 64.53%, respectively) than the Q-Sepharose Kit (28.11 and 38.69%, respectively). Correction of the cfDNA yield with either the extraction efficiency of the 89 or 180 bp fragment of CEREBIS decreased the variance when extraction was with Q-sepharose, but not the Zymo, Kit. However, correction using creatinine provided the greatest reduction in variability regardless of the extraction method.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Biospecimen Aliquots and Components	Aliquot size/volume	50 mL 10 mL 20 mL 30 mL 40 mL
   Analyte Extraction and Purification	Analyte isolation method	Zymo Kit Q-sepharose Isolated by three different researchers Three extractions by each researcher
   Digital PCR Specific	Length of gene fragment	89 bp CEREBIS spike-in 180 bp CEREBIS Spike-in
   Digital PCR Specific	Data handling	Corrected for CEREBIS 89 bp Corrected for CEREBIS 180 bp Corrected for creatinine

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2. Study Purpose

   This study investigated the source of variability in cfDNA extraction from the plasma of healthy men by comparing the variability associated among specimens, researchers performing the extraction, extractions performed by one researcher on different days, and among assays from the same extraction. Blood was collected from nine healthy men into Sarstedt S-Monovette K₃EDTA tubes. Plasma was separated by centrifugation at 2000 g for 15 min followed by 3800 g for 10 min. Plasma was stored at -80°C. Plasma from six men was pooled and aliquoted, and cfDNA extraction was performed in triplicate on three separate days by three different researchers and then assayed in triplicate. To compare intra- versus inter-individual variability, cfDNA was extracted from the remaining three plasma specimens in triplicate and then assayed in triplicate. Prior to cfDNA extraction with the QIAamp Circulating Nucleic Acid Kit, plasma was spiked with 20,000 copies of a 180 bp fragment of CEREBIS. cfDNA was quantified using the Qubit 1X dsDNA HS Assay Kit and by droplet digital PCR (ddPCR) amplification of AGO1, SRY and RPP30. The size distribution of the isolated cfDNA was analyzed using the High Sensitivity DNA Kit with the 2100 Bioanalyzer system.

   Summary of Findings:

   The size distribution pattern of cfDNA extracted from plasma using the QIAamp Circulating Nucleic Acid Kit was consistent among specimens, with the expected peak at 170 bp. Notably, the cfDNA pattern in plasma was distinct from that in urine. When cfDNA was extracted from triplicate aliquots of pooled plasma (from five men) by three different researchers on three different days and quantified by ddPCR in triplicate. The largest variance in all three assays was within a single extraction (assay variability, 46.09-66.86%), followed by between extractions by the same researcher on the same day (23.45- 28.48%), and extractions by the same researcher on different days (9.70-25.63%), with the least variability attributed to the researcher performing the extraction (0-4.53%). When urine from 3 men was spiked with a 180 bp fragment of CEREBIS and then extracted in triplicate, 84.55% of the variability was found to be inter-individual, with 6.76% and 8.68% of the variability attributed to intra- (assay) and inter- extraction, respectively. When the cfDNA copies were corrected for extraction efficiency using the spike-in control (180 bp CEREBIS), the variability in the assay was reduced, and the inter-extraction variability in SRY and RPP30, but not AGO1, was reduced.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Isolated by three different researchers Three extractions by each researcher on different days
   Digital PCR Specific	Targeted nucleic acid	SRY RPP30 AGO1
   Digital PCR Specific	Data handling	Correction for CEREBIS 180 bp Uncorrected

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