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Degradation of methylation signals in cryopreserved DNA.

Author(s): Lee NY, Hum M, Tan GP, Seah AC, Kin PT, Tan NC, Law HY, Lee ASG

Publication: Clin Epigenetics, 2023, Vol. 15, Page 147

PubMed ID: 37697422 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of the study was to compare DNA methylation before and after storage of extracted DNA at -20°C for up to 50 months.  Peripheral blood was collected from a total of 262 volunteers of Chinese ethnicity without cancer as part of a discovery (126 volunteers) and validation (136 volunteers) cohort.  Buffy coat was isolated from blood specimens in the discovery cohort within 2 h and was subsequently stored at -20°C for up to 3 months (median 20 days) before DNA extraction. For the validation cohort, blood was stored at 4°C for up to 5 days before DNA extraction. DNA was isolated using the QIAamp DNA Blood Kit. DNA was extracted from specimens in the discovery cohort using the QIAamp DNA Blood Kit and then stored at -20°C for 33.5-50.1 months (median 44.4 months). In contrast, DNA was extracted from specimens in the validation cohort using the MagNA Pure Compact System and stored at 4°C for up to 2 months and then at -20°C for a total storage duration of 15.0-38.0 months (median 21 months). DNA was quantified using the QuantiFluor dsDNA system and a NanoDrop spectrophotometer. DNA was bisulfite converted using the EZ DNA Methylation Kit,  and methylation was profiled on the Infinium Methylation EPIC bead chip.

   Summary of Findings:

   Mean global methylation was not affected when extracted DNA was stored at -20°C  for up to 50 months even after controlling for patient age, blood cell type composition, and buffy coat storage duration in specimens from the discovery cohort. In the validation cohort, there was an increase in mean global methylation with progressive storage of extracted DNA at -20°C  (P=0.03), although the increase was not significant after correcting for patient age and blood cell type composition. Further, when methylation β-values were used (instead of M-values) there was no association between methylation and storage in either cohort. Blood cell type composition was associated with global methylation (P≤ 4.0e-08).

   Examination of individual CpGs revealed a trend toward hypomethylation with progressive storage at -20°C in both cohorts.  In the discovery cohort, 4,049 of the 830,545 CpGs analyzed were hypomethylated, but only 50 CpGs were hypermethylated after -20°C storage. After correction for patient age, cell type composition, and storage duration, 18,311 hypomethylated CpGs and 21 hypermethylated CpGs were found. In the validation cohort, 63 CpGs were hypomethylated and 6 were hypermethylated after frozen storage of DNA, although only 3 hypomethylated CpGs remained significant after correction for covariates.  The authors stated that the smaller number of hypomethylated CpGs that were observed after storage in the validation cohort could be explained by either the shorter storage duration or the fact that the storage period included up to two months at 4°C for that cohort. The authors stated that there was more overlap in the list of hypomethylated CpGs after -20°C storage of DNA between the two cohorts than would be expected by chance. Further analysis revealed that CpGs close to CpG islands were more likely to be hypomethylated and those located within CpG islands were less likely to be hypomethylated.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	33.5-50.1 months 15.0-38.0 months
   Storage	Storage conditions	As buffy coat/blood As extracted DNA

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