title: Method Validation for Extraction of Nucleic Acids from Peripheral Whole Blood.
abstract: BACKGROUND
  This article is the fifth in a series of publications providing formal method validation for biospecimen processing. We report the optimization and validation of methodology to obtain nucleic acids of sufficient quantity and quality from blood.
  
  
  METHODS
  DNA was extracted using the Chemagic DNA Blood Kit on an MSM I. Extraction was optimized in terms of blood volume, elution buffer volume, and lysis conditions. The optimal protocol was validated for reproducibility, robustness (delay to buffy coat extraction, blood vs. buffy coat, and use of a magnetic rack), and performance (yield, purity, and concentration). RNA was extracted using a PAXgene Blood miRNA kit with a QiaCube. The protocol was validated for reproducibility, robustness (elution buffer, delay, and temperature before extraction), and performance (yield, purity, integrity, and miRNA content). Two platforms (QiaCube, Biorobot Universal) were further compared.
  
  
  RESULTS
  For DNA extraction, a 4 mL blood sample, manual lysis, and 300 μL elution buffer were found to be reproducible (CV <10% for DNA yield and A260 nm/A280 nm ratio) and robust (buffy coat vs. whole blood; immediate processing of buffy coat after lysis vs. storage for 1 week at 2-8°C; and magnetic rack use). There was no difference between automated and manual lysis. RNA extracted with the PAXgene Blood miRNA kit on a QiaCube gave high yields and optimal reproducibility (low CV for RNA yield and integrity) with BR5 elution buffer (vs. water and TE). PAXgene tubes could be stored for up to 2 weeks at 2-8°C. The Biorobot Universal System gave similar mean RNA yields with Qiacube and slightly lower but acceptable purity.
  
  
  CONCLUSIONS
  We validated automated isolation of DNA with a Chemagic DNA Blood Kit on a magnetic bead-based MSM I, and of RNA with a PAXgene Blood miRNA kit on a silica membrane-based QiaCube or Biorobot (for low and high throughput, respectively).
authors.affiliation: Integrated BioBank of Luxembourg (IBBL) , Luxembourg City, Luxembourg .
authors.forename: Conny
authors.initials: C
authors.lastname: Mathay
authors.affiliation: Integrated BioBank of Luxembourg (IBBL) , Luxembourg City, Luxembourg .
authors.forename: Gaël
authors.initials: G
authors.lastname: Hamot
authors.affiliation: Integrated BioBank of Luxembourg (IBBL) , Luxembourg City, Luxembourg .
authors.forename: Estelle
authors.initials: E
authors.lastname: Henry
authors.affiliation: Integrated BioBank of Luxembourg (IBBL) , Luxembourg City, Luxembourg .
authors.forename: Kathleen
authors.initials: K
authors.lastname: Mommaerts
authors.affiliation: Integrated BioBank of Luxembourg (IBBL) , Luxembourg City, Luxembourg .
authors.forename: Audur
authors.initials: A
authors.lastname: Thorlaksdottir
authors.affiliation: Integrated BioBank of Luxembourg (IBBL) , Luxembourg City, Luxembourg .
authors.forename: Johanna
authors.initials: J
authors.lastname: Trouet
authors.affiliation: Integrated BioBank of Luxembourg (IBBL) , Luxembourg City, Luxembourg .
authors.forename: Fay
authors.initials: F
authors.lastname: Betsou
mesh_terms: D057205:Automation, Laboratory; D001800:Blood Specimen Collection / Q000379:methods*; D004247:DNA / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D012313:RNA / Q000097:blood / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results
chemical_list: D011933:Reagent Kits, Diagnostic; D012313:RNA; D004247:DNA
keywords: DNA; RNA; method validation; processing method; whole blood