Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

Ghantous, Akram; Saffery, Richard; Cros, Marie-Pierre; Ponsonby, Anne-Louise; Hirschfeld, Steven; Kasten, Carol; Dwyer, Terence; Herceg, Zdenko; Hernandez-Vargas, Hector

doi:10.1186/1472-6750-14-60

BMC Biotechnology

Table 6 Cross-comparisons of DNA quantity and quality parameters among the different tested DNA extraction protocols

From: Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

Protocol	DNA quantity (ng)^a	260/280 ratio (after heating)^b	260/230 ratio (after heating)^c	DNA integrity^d	Detectability by PCR
GQ	808 ± 376 ng	1.83 ± 0.14	0.92 ± 0.23	Peak intensity > 1 Kbp (25/32)	Detectable (42/42)
QQ	Lower (3/3) 175 ± 73 ng	Similar (2/3); Worse (1/3)	Lower (3/3)	NA^e	Similar (3/3)
Qq	Higher (6/6) by 2.0 ×	Worse (6/6) 0.89 ± 0.22	Similar (6/6)	Similar (4/6); Better (1/6); Worse (1/6)	Similar (6/6)
GN or Gn	Higher (4/6) by 1.5 ×; Similar (2/6)	Similar (6/6)	Similar (4/6); Higher (2/6)	Worse (4/4)	Similar (6/6)
*GN-XS* or *Gn-XS*	Higher (6/9) by 1.7 ×; Similar (2/9); Lower (1/9) by 0.4 ×	Similar (6/9); Worse (3/9)	Similar (5/9); Lower (4/9)	Worse (6/6)	Similar (9/9)
NN or Nn	Higher (8/9) by 1.7 ×; Similar (1/9)	Similar (9/9)	Higher (9/9) 1.71 ± 0.20	Worse (6/6)	Similar (9/9)
*NN-XS*	Similar (2/6); Higher (2/6) by 1.8 ×; Lower (2/6) by 0.3 ×	Similar (5/6); Worse (1/6)	Similar (6/6)	Worse (3/5); Similar (1/5); Better (1/5)	Similar (6/6)

The GQ protocol is set as the reference protocol above all the other protocols to which it is compared. The DNA quantity or quality parameters of the other protocols are described always in comparison to GQ. DNA yields of QQ were significantly lower than GQ (p < 0.05), those of Qq, GN, Gn, NN and Nn were significantly higher than GQ (p < 0.05), and those of GN-XS, Gn-XS and NN-XS were not different from GQ (p > 0.05), as compared by paired t-test. For each DNA parameter described, the counts of hits over the total number of DBS analyzed for that parameter are indicated between parentheses. Table cells highlighted in bold represent protocol performance that is at least as good as that of GQ.
^aQuantities showing less than 20% change from GQ were considered ‘similar’ to GQ. This threshold exceeds the 15.7% average increase in DNA quantities observed between GQ technical replicates, with an average coefficient of variation of 12.3% (n = 6 pairs of replicates).
^bIn protocol pairwise comparisons, 260/280 ratios that were considered ‘similar’ were either both within the optimal 1.7-2.0 range or both outside this range. Otherwise, the ratio outside the 1.7-2.0 range was considered ‘worse’ relative to that within.
^c‘Lower’ and ‘higher’ indicate 260/230 ratio differences of at least 0.30 absorbance units below or above GQ ratios, respectively; otherwise, ratios were considered ‘similar’. The following guidelines were adopted for the 260/230 ratio: optimal and indicating pure DNA if higher than 2.0, acceptable if between 1.5-2.0, and tolerated if between 1.0-1.49 (Macherey-Nagel GmbH & co. KG, reference 740230).
^dDNA integrity refers to DNA size range and level of degradation, as assessed by gel electrophoresis. Ten samples were also reassessed by bioanalyzer, showing similar relative comparisons to GQ. Every DBS, in which both punches of the tested protocol pairs exhibited high DNA degradation (size range below 1 Kbp), was excluded from the pairwise comparisons of protocols.
^eNA: Not Applicable due to limited quantities of extractable DNA.

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