A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification
| Autor: | Daniel R A Cox, Boris Ka Leong Wong, Su Kah Goh, Alexander Dobrovic, Hongdo Do, Tom Witkowski, Ashan Musafer, Vijayaragavan Muralidharan |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Chromatography Chemistry Biochemistry (medical) Clinical Biochemistry Extraction (chemistry) Reproducibility of Results DNA DNA extraction Bisulfite Liver transplant recipient 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Cell-free fetal DNA Synthetic DNA 030220 oncology & carcinogenesis DNA methylation Humans Sulfites Digital polymerase chain reaction Cell-Free Nucleic Acids |
| Zdroj: | Clinical chemistry. 67(9) |
| ISSN: | 1530-8561 |
| Popis: | Background Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. Methods We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. Results Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. Conclusions CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary. |
| Databáze: | OpenAIRE |
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