Abstract
BackgroundNeonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses.ResultsUsing combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent.ConclusionsThis study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS.
Highlights
Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin
Optimization of Phases I and II in the DNA extraction protocols Limited quantity and quality are important drawbacks in the use of DNA obtained from DBS, for epigenome-wide studies
We ruled out the possibility of using whole bisulfitome amplification (WGA) after confirming the introduction of biases, mostly in the middle range of DNA methylation levels (Additional file 1), further confirming the recently reported finding by Bundo et al [13]
Summary
Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. It is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses Epigenetic mechanisms, such as DNA methylation, have been suggested as possible causal pathways linking environmental exposure to disease. Many of these studies depend on the epigenome-wide analysis of prospectively collected samples, in the context of large human cohorts. Flinders Technology Associates chemically treated filter papers (FTA cards) were developed for DNA/RNA analyses [4] These chemically treated cards allow long-term storage of DNA at room temperature and are impregnated with denaturants that guard against oxidation, nuclease and ultraviolet damage, and both bacterial and fungal degradation
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