Differences in amplification efficiency of standard curves in quantitative real-time PCR assays and consequences for gene quantification in environmental samples.
High and comparable efficiency values are the key for reliable quantification of target genes from environmental samples using real-time PCR. Therefore it was the aim of this study to investigate if PCR amplification efficiencies of plasmid DNA used for the calculation of standard curves (i) remain constant along a logarithmic scale of dilutions and (ii) if these values are comparable to those of DNA extracted from environmental samples. It could be shown that comparable efficiency values within the standards cannot be achieved using log scale serial dilutions and a comparison of gene copy numbers from DNA extracted from environmental samples and standard DNA extracted from plasmids is only possible in a very small interval.
Töwe
Stefanie
S
Technische Universität München, Chair of Soil Ecology, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. stefanie.toewe@helmholtz-muenchen.de
Kleineidam
Kristina
K
Schloter
Michael
M
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004783:Environmental Microbiology; D018628:Gene Dosage; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards
D001426:Bacterial Proteins; D004269:DNA, Bacterial