Comparison of UV spectrometry and fluorometry-based methods for quantification of cell-free DNA in red cell components.
Author(s): Khetan D, Gupta N, Chaudhary R, Shukla JS
Publication: Asian J Transfus Sci, 2019, Vol. 13, Page 95-99
PubMed ID: 31896914 PubMed Review Paper? No
Purpose of Paper
This paper compared spectrophotometry and fluorometry quantification of cell-free DNA (cfDNA) isolated from red blood cell (RBC) supernatants.
Conclusion of Paper
cfDNA was quantifiable by both methods in 92 of the specimens but were too low for quantification by either method in the remaining seven specimens. The median measured cfDNA concentration was higher when measured by NanoDrop than when measured by Qubit and measured concentrations were only very weakly correlated between the two methods.
Studies
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Study Purpose
This study compared spectrophotometry and fluorometry quantification of cfDNA isolated from RBC supernatants. Whole blood was collected from 99 blood donors and packed RBCs (PRBCs) were obtained by the buffy coat-depletion method using Cryofuge 6000i and a Terumo Automatic Component Extractor–II. PRBCs were stored in the refrigerator for 21 days. After storage, PRBCs were centrifuged at 1050 rpm for 9 min at 4°C followed by 3000 rpm for 5 min and then the supernatant was collected and stored at or below −40°C. DNA was extracted using the QIAamp DNA Blood Mini Kit and quantified by a Nanodrop spectrophotometer and a Qubit fluorometer using the Qubit dsDNA HS Assay Kit.
Summary of Findings:
Although DNA quantification was generally very close to the estimated concentration for both methods when using a known standard at low dilutions, only Qubit was found to be both precise and accurate when DNA was highly diluted (1.35–0.16 ng/μl). cfDNA was quantifiable by both methods in 92 of the 99 specimens but were too low for quantification by either method in the remaining seven specimens. The median measured cfDNA concentration was higher when measured by NanoDrop than when measured by Qubit (1.60 ng/μl versus 0.80 ng/μl, P<0.05). Further, measured concentrations were only very weakly correlated between the two methods (R2=0.021, significance not determined).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
- Normal
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Spectrophotometry Specific Technology platform NanoDrop
Qubit dsDNA HS Assay Kit