Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience.
Author(s): Malentacchi F, Pizzamiglio S, Verderio P, Pazzagli M, Orlando C, Ciniselli CM, Günther K, Gelmini S
Publication: Clin Chem Lab Med, 2015, Vol. , Page
PubMed ID: 25883202 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of DNA extraction method and delayed isolation on the yield of cell-free DNA. Inter-laboratory variability was also examined.
Conclusion of Paper
The integrity of cell-free DNA was variable among the 56 participating laboratories. While no effect of storing plasma beyond the recommended 3 days at 4ºC was identified, use of a cell-free DNA-specific DNA extraction kit rather than a general DNA extraction kit resulted in more intact DNA when extraction was performed within 3 days.
Studies
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Study Purpose
The purpose of this study was to determine the effect of DNA extraction method and delayed isolation on the yield of cell-free DNA. Inter-laboratory variability was also examined. Plasma from a single healthy donor was obtained by double centrifugation, aliquoted, stored at 4ºC and shipped at 2-8ºC. After arrival, specimens were stored for <3 days at 4ºC before DNA extraction. Extracted DNA was then shipped back at 4ºC. DNA integrity was determined by the multiplex PCR-based DNA Quality Check Kit. Importantly, the analysis of plasma storage and extraction kit only included the 50 laboratories that reported the plasma having arrived at the appropriate temperature and in the appropriate condition.
Summary of Findings:
Of the 56 participating laboratories, 4 (7%) failed to isolate amplifiable cell-free DNA, 20 (36%) isolated DNA of low integrity (100-200 bp maximum length), 25 (44.6%) isolated DNA of intermediate integrity and 7 (12.5%) isolated high integrity DNA (600 bp maximum length). There was no significant difference in RNA integrity among specimens processed at the 44 laboratories that stored plasma for <3 days at 4ºC and the 6 that stored plasma at -80ºC or 4ºC for up to 12 days. When extraction was performed within 3 days of blood collection, kits tailored to cell-free DNA generated more intact DNA than general DNA extractions kits; however, no such difference among extraction kits were observed when all delays and laboratories were compared.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method General DNA extraction kit
Cell-free DNA extraction kit
Storage Storage duration <3 days
3-12 days
Storage Storage temperature 4ºC
-80ºC or 4ºC
PCR Specific Length of gene fragment 100 bp
200 bp
300 bp
400 bp
500 bp
600 bp