Effects of Repeated Freeze and Thaw Cycles on the Genome-Wide DNA Methylation Profile of Isolated Genomic DNA.
Author(s): Kopfnagel V, Klopp N, Bernemann I, Nizhegorodtseva N, Wilson R, Gronauer R, Seifert M, Illig T
Publication: Biopreserv Biobank, 2023, Vol. , Page
PubMed ID: 37074140 PubMed Review Paper? No
Purpose of Paper
This paper compared the methylation profile of genomic DNA (gDNA) subjected to 0, 1, 3, 5 or 10 freeze-thaw cycles.
Conclusion of Paper
No differentially methylated probes (<0.1 false discovery rate, P<0.05) were found when specimens subjected to 0 and 10 freeze-thaw cycles were compared. Further, specimens clustered in Multidimensional Scaling (MDS) plots and dendrograms based on patient source not the number of freeze-thaw cycles and β-values were not significantly affected by the number of freeze-thaw cycles.
Studies
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Study Purpose
This study compared the methylation profile of gDNA subjected to 0, 1, 3, 5 or 10 freeze-thaw cycles. EDTA blood from nine healthy men and ten healthy women was centrifuged at 2000 g for 10 min immediately after collection. The buffy coat was automatically detected and aliquoted using a Hamilton easyBlood Workstation and stored at -80℃. DNA was extracted using the STAR DNA-Blood1k Kit and a Hamilton Chemagic STAR Automated Sample Preparation System. DNA was quantified using a NanoDrop spectrometer. Isolated DNA was aliquoted and matched aliquots were frozen and stored continuously at -80℃ for 10 months or stored at -80℃ with 1, 3, 5 or 10 monthly freeze-thaw cycles (at room temperature for 6 h followed by refreezing in a -80℃ freezer). Methylation levels were assessed using Illumina Infinium MethylationEPIC BeadChip assays in DNA stored frozen for 10 months (0 cycles), and in DNA freeze-thaw cycled 1, 3, 5 or 10 times over a 10 month period.
Summary of Findings:
β-values from all specimens displayed the expected distribution and, when graphed together, the lines overlapped regardless of the number of freeze-thaw cycles. Multidimensional Scaling (MDS) plots based on the 1,000 most variable CpG sites clustered specimens based on the patient (not number of freeze-thaw cycles), regardless of whether the date used was raw or normalized. Similarly, in dendrograms created with ChAMP, clustering was by patient source and not number of freeze-thaw cycles. ANOVA analysis found no significant differences among β-values from specimens subjected to different numbers of freeze-thaw cycles and no differentially methylated probes (<0.1 false discovery rate, P<0.05) were found between specimens subjected to 0 or 10 freeze-thaw cycles.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA DNA microarray DNA Spectrophotometry DNA Bisulfite conversion assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 0 cycles
1 cycle
3 cycles
5 cycles
10 cycles