Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR.
OBJECTIVE
The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae.


RESULTS
Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots.


CONCLUSIONS
Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation.


CONCLUSIONS
This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.
Bushon
R N
RN
Ohio Water Microbiology Laboratory, Ohio Water Science Center, U.S. Geological Survey, Columbus, OH 43229-1111, USA. rnbushon@usgs.gov
Kephart
C M
CM
Koltun
G F
GF
Francy
D S
DS
Schaefer
F W
FW
Alan Lindquist
H D
HD
D001408:Bacillus anthracis / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D005604:Francisella tularensis / Q000235:genetics*; D007202:Indicators and Reagents / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011933:Reagent Kits, Diagnostic / Q000592:standards / Q000706:statistics & numerical data*; D016019:Survival Analysis; D014734:Vibrio cholerae / Q000235:genetics*
D004269:DNA, Bacterial; D007202:Indicators and Reagents; D011933:Reagent Kits, Diagnostic