Optimized DNA extraction from neonatal dried blood spots: application in methylome profilingReport as inadecuate




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BMC Biotechnology

, 14:60

Genome technology and molecular diagnostics

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.

KeywordsBlood spot DNA extraction Epigenetics Methylome HM450 Pyrosequencing Whole bisulfitome amplification QIAamp GenSolve NucleoSpin Abbreviationsbpbase pair

DBSdried blood spots

gDNAgenomic DNA

GQGenSolve-QIAamp

GNGenSolve-NucleoSpin

GnGN with NucleoSpin’s precipitation buffer replaced by ethanol

GN-XSGN extra small

Gn-XSGN-XS with NucleoSpin’s precipitation buffer replaced by ethanol

HM450Infinium HumanMethylation450 Beadchip

Kbpkilo base pair

NCSNational Children Study - USA

NNNucleoSpin-NucleoSpin

NnNN with NucleoSpin’s precipitation buffer replaced by ethanol

NN-XSNN extra small

QCquality control

QQQIAamp-QIAamp

QqQQ with QIAamp’s precipitation buffer replaced by ethanol

SWANSubset-quantile Within Array Normalisation

TIHSTasmanian Infant Health Survey - Australia.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-14-60 contains supplementary material, which is available to authorized users.

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Author: Akram Ghantous - Richard Saffery - Marie-Pierre Cros - Anne-Louise Ponsonby - Steven Hirschfeld - Carol Kasten - Terence Dw

Source: https://link.springer.com/







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