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Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling
- 作者:Akram Ghantous (1)
Richard Saffery (2) Marie-Pierre Cros (1) Anne-Louise Ponsonby (3) (4) Steven Hirschfeld (5) Carol Kasten (5) Terence Dwyer (3) (4) (6) Zdenko Herceg (1) Hector Hernandez-Vargas (1)
1. Epigenetics Group ; International Agency for Research on Cancer (IARC) ; 150 rue Albert-Thomas ; 69008 ; Lyon ; France 2. Cancer and Disease Epigenetics ; Murdoch Childrens Research Institute Royal Children鈥檚 Hospital ; Flemington Road ; Parkville ; Victoria ; 3052 ; Australia 3. Environmental & Genetic Epidemiology Research ; Murdoch Children鈥檚 Research Institute Royal Children鈥檚 Hospital ; Flemington Road ; Parkville ; Victoria ; 3052 ; Australia 4. Menzies Research Institute ; University of Tasmania ; Hobart ; TAS ; 7000 ; Australia 5. Department of Health and Human Services ; National Children鈥檚 Study ; Eunice Kennedy Shriver National Institute of Child Health and Human Development ; National Institutes of Health U.S. ; 6100 Executive Boulevard ; Room 3A01 ; Bethesda ; MD ; 20892 ; USA 6. Chair ; Steering Committtee ; International Childhood Cancer Cohort Consortium (I4C) ; Bethesda ; USA
- 关键词:Blood spot ; DNA extraction ; Epigenetics ; Methylome ; HM450 ; Pyrosequencing ; Whole bisulfitome amplification ; QIAamp ; GenSolve ; NucleoSpin
- 刊名:BMC Biotechnology
- 出版年:2014
- 出版时间:December 2014
- 年:2014
- 卷:14
- 期:1
- 全文大小:1,177 KB
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- 刊物类别:Chemistry and Materials Science
- 刊物主题:Biotechnology
Life Sciences Plant Breeding/Biotechnology Stem Cells Transgenics
- 出版者:BioMed Central
- ISSN:1472-6750
文摘
Background 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. 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. Results Using 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. Conclusions This 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.
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