Bars = estimated 16S copies based on nicked-circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon-based standard. Data are the average (n = 3) and error bars are 61 standard deviation among replicates. doi:10.1371/journal.pone.0051931.gNb.BtsI digest, SpeI digest, and end-point PCR, respectively. The four DNA preparations were purified, quantified using Qubit fluorometry, and analyzed by agarose gel electrophoresis (Figure 1). Propagated plasmids isolated from transformed PF-299804 web Bacterial cells were predominantly supercoiled DNAs that ran faster than their linearized counterparts (Figure 1a, compare lanes labeled S to lanes L), whereas the nicked circles ran much slower than both the linearized and supercoiled plasmids. The 16S rRNA gene amplicons that spanned the V1 2 region were approximately 350 base pairs in length (Figure 1b.). Next, to determine if the conformation of the DNA standard significantly affected amplification efficiency, the performance of qPCR reactions using serial dilutions of the four prepared standards were compared (Figure 2). Bacterial T. lienii curves spanned from 107 to 103 copies (Figure 2a and Table 2) and the performance of each standard curve is summarized in Table 3. Amplification efficiencies ranged from 85 to 89 , and an ANOVA showed that there was no significant difference between the slopes or y-intercepts of the four curves (P = 0.97). Similar results were obtained for the A. fulgidus 16S rRNA gene standards (Figure 2b and Table 2 and Table 3).Amplification efficiencies ranged from 88 to 94 and the four curves were not significantly different from one another (P = 0.99) by ANOVA. Therefore, the conformation of the standard had a negligible effect on the performance of the qPCR reactions. These results were not unexpected, as the efficiencies were not consistently different for eukaryotic gene amplification [7].Comparison of Microbial 16S rRNA Gene Copies Based on Standard CurvesWhile Hou et al. [7] found no consistent difference between amplification efficiencies between circular and linear curves, they did however find that standard curves based on the circular plasmids overestimated the number of gene copies in their eukaryotic system by approximately 8-fold. Therefore, using two bacterial and two archaeal genomes we asked if either circular plasmid conformation caused the same degree of inflation. MedChemExpress CTX-0294885 Genomic DNA samples were assayed at three dilutions: 1:10, 1:50, and 1:100, each in triplicate. This range was deemed appropriate as DNA extracted from environmental samples mayEffect of qPCR Standards on 16S Gene EstimatesFigure 4. Comparison of expected and estimated 16S rRNA gene copies in archaeal DNA samples. Expected archaeal 16S rRNA gene copies were calculated based on one and two 16S copies per genome for (a) A. fulgidus and (b) M. jannaschii, respectively. Black bars = predicted 16S copies. White bars = estimated 16S copies based on supercoiled plasmid standard. Grey bars = estimated 16S copies based on nicked circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon standard. 18325633 Data shown are representative of two experiments. Data are the average (n = 3) and error bars are 61 standard deviation among replicates. doi:10.1371/journal.pone.00519.Bars = estimated 16S copies based on nicked-circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon-based standard. Data are the average (n = 3) and error bars are 61 standard deviation among replicates. doi:10.1371/journal.pone.0051931.gNb.BtsI digest, SpeI digest, and end-point PCR, respectively. The four DNA preparations were purified, quantified using Qubit fluorometry, and analyzed by agarose gel electrophoresis (Figure 1). Propagated plasmids isolated from transformed bacterial cells were predominantly supercoiled DNAs that ran faster than their linearized counterparts (Figure 1a, compare lanes labeled S to lanes L), whereas the nicked circles ran much slower than both the linearized and supercoiled plasmids. The 16S rRNA gene amplicons that spanned the V1 2 region were approximately 350 base pairs in length (Figure 1b.). Next, to determine if the conformation of the DNA standard significantly affected amplification efficiency, the performance of qPCR reactions using serial dilutions of the four prepared standards were compared (Figure 2). Bacterial T. lienii curves spanned from 107 to 103 copies (Figure 2a and Table 2) and the performance of each standard curve is summarized in Table 3. Amplification efficiencies ranged from 85 to 89 , and an ANOVA showed that there was no significant difference between the slopes or y-intercepts of the four curves (P = 0.97). Similar results were obtained for the A. fulgidus 16S rRNA gene standards (Figure 2b and Table 2 and Table 3).Amplification efficiencies ranged from 88 to 94 and the four curves were not significantly different from one another (P = 0.99) by ANOVA. Therefore, the conformation of the standard had a negligible effect on the performance of the qPCR reactions. These results were not unexpected, as the efficiencies were not consistently different for eukaryotic gene amplification [7].Comparison of Microbial 16S rRNA Gene Copies Based on Standard CurvesWhile Hou et al. [7] found no consistent difference between amplification efficiencies between circular and linear curves, they did however find that standard curves based on the circular plasmids overestimated the number of gene copies in their eukaryotic system by approximately 8-fold. Therefore, using two bacterial and two archaeal genomes we asked if either circular plasmid conformation caused the same degree of inflation. Genomic DNA samples were assayed at three dilutions: 1:10, 1:50, and 1:100, each in triplicate. This range was deemed appropriate as DNA extracted from environmental samples mayEffect of qPCR Standards on 16S Gene EstimatesFigure 4. Comparison of expected and estimated 16S rRNA gene copies in archaeal DNA samples. Expected archaeal 16S rRNA gene copies were calculated based on one and two 16S copies per genome for (a) A. fulgidus and (b) M. jannaschii, respectively. Black bars = predicted 16S copies. White bars = estimated 16S copies based on supercoiled plasmid standard. Grey bars = estimated 16S copies based on nicked circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon standard. 18325633 Data shown are representative of two experiments. Data are the average (n = 3) and error bars are 61 standard deviation among replicates. doi:10.1371/journal.pone.00519.
