Ilar overestimates using representatives from microbial domains. We demonstrated that estimates of 16S rRNA gene copies did not approach the 8-fold overestimates reported for eukaryotic systems [7]. Indeed, estimates derived from the supercoiled standardEffect of qPCR Standards on 16S Gene EstimatesTable 4. Estimated and expected 16S rRNA gene copies in microbial gDNA samples based on qPCR standard curves.Ratioa 0.8460.06 1.4260.51 1.4860.52 2.1760.Genome P. aeruginosaType Amplicon Linear Nicked circles Supercoiled Predictedb1:10 8.0160.116105 1.3960.1:50 1.5160.126105 2.6260.1:100 8.0860.486104 1.4160.086105 1.3960.086105 2.0060.126105 1.0160.086105 1.3360.066105 2.3160.116105 2.3060.146105 3.3460.1.4460.026106 2.1460.036106 8.8760.106105 1.3660.096106 2.3660.166106 2.4860.176106 3.7060.2.6260.226105 3.8060.326105 1.8460.MedChemExpress Biotin NHS 016105 2.6160.026105 4.5360.036105 4.5960.036105 6.7160.D. vulgarisAmplicon Linear Nicked circles Supercoiled Predicted0.5060.19 0.8760.34 0.8960.35 1.3060.2.1560.04×6106 7.0460.766105 8.8760.4.6060.046105 1.4460.136105 1.8060.2.5860.016105 6.1160.396104 7.6060.496104 7.8660.ML-281 web 536104 8.7260.566104 1.2860.026105 7.9260.366104 9.8660.456104 1.0360.A. fulgidusAmplicon Linear Nicked circles Supercoiled Predicted0.5060.04 0.6360.06 0.6860.08 0.7460.1.0160.116106 1.0660.116106 1.3660.056106 8.1360.516105 1.0360.066106 1.1760.1.9260.186105 2.1060.206105 2.8060.016105 1.7360.046105 2.1660.046105 2.3360.M. jannaschiiAmplicon Linear Nicked circles Supercoiled Predicted0.7160.03 0.8960.04 0.9660.04 1.0460.1.2360.086106 1.1960.2.5260.066105 2.4160.1.1460.056105 1.1060.abRatio of estimated divided by predicted 16S copies averaged across the 16574785 three dilutions. Predicted copies calculated as described in Methods. doi:10.1371/journal.pone.0051931.tcurves ranged from 0.5 to 2.2-fold and no single conformation provided the best estimates for the genomes tested. Aside from the conformation of the DNA target gene, several variables between the three studies could account in part for the differences in magnitude of gene estimates observed between eukaryotic versus prokaryotic systems. Those include but are not limited to: 1) the conformation of the circular standard tested and 2) the preparation of the standards. In the Hou et al. study [7], the implication that the circular plasmid was supercoiled must be inferred from the text, as a gel image was not included. In any case, estimates were much higher than those 
using the linear standard. In the maize study [8], 3-fold inflation in gene estimates was observed for the supercoiled versus the linearized standard. Interestingly, both supercoiled and nicked 24195657 circular plasmids were prepared, but only the supercoiled was investigated for its affect on estimates using genomic DNA [8]. In results presented here, the effect of both circular plasmid conformations were assayed using microbial genomic DNA. Another source of variation was the method of standard preparation and quantification. Hou et al. purified the plasmids and amplicons prior to quantification based on the optical absorbance at OD260 [7]. Lin et al. demonstrated that supercoiled and linearized DNA showed differences in quantification based on the optical absorbance, but took measurements prior to purification [8]. In the present study, the digested plasmid standards and amplicons were purified andquantified following digestion or amplification to rid of enzymes or other contaminants that could interfere with quantification readings or com.Ilar overestimates using representatives from microbial domains. We demonstrated that estimates of 16S rRNA gene copies did not approach the 8-fold overestimates reported for eukaryotic systems [7]. Indeed, estimates derived from the supercoiled standardEffect of qPCR Standards on 16S Gene EstimatesTable 4. Estimated and expected 16S rRNA gene copies 
in microbial gDNA samples based on qPCR standard curves.Ratioa 0.8460.06 1.4260.51 1.4860.52 2.1760.Genome P. aeruginosaType Amplicon Linear Nicked circles Supercoiled Predictedb1:10 8.0160.116105 1.3960.1:50 1.5160.126105 2.6260.1:100 8.0860.486104 1.4160.086105 1.3960.086105 2.0060.126105 1.0160.086105 1.3360.066105 2.3160.116105 2.3060.146105 3.3460.1.4460.026106 2.1460.036106 8.8760.106105 1.3660.096106 2.3660.166106 2.4860.176106 3.7060.2.6260.226105 3.8060.326105 1.8460.016105 2.6160.026105 4.5360.036105 4.5960.036105 6.7160.D. vulgarisAmplicon Linear Nicked circles Supercoiled Predicted0.5060.19 0.8760.34 0.8960.35 1.3060.2.1560.04×6106 7.0460.766105 8.8760.4.6060.046105 1.4460.136105 1.8060.2.5860.016105 6.1160.396104 7.6060.496104 7.8660.536104 8.7260.566104 1.2860.026105 7.9260.366104 9.8660.456104 1.0360.A. fulgidusAmplicon Linear Nicked circles Supercoiled Predicted0.5060.04 0.6360.06 0.6860.08 0.7460.1.0160.116106 1.0660.116106 1.3660.056106 8.1360.516105 1.0360.066106 1.1760.1.9260.186105 2.1060.206105 2.8060.016105 1.7360.046105 2.1660.046105 2.3360.M. jannaschiiAmplicon Linear Nicked circles Supercoiled Predicted0.7160.03 0.8960.04 0.9660.04 1.0460.1.2360.086106 1.1960.2.5260.066105 2.4160.1.1460.056105 1.1060.abRatio of estimated divided by predicted 16S copies averaged across the 16574785 three dilutions. Predicted copies calculated as described in Methods. doi:10.1371/journal.pone.0051931.tcurves ranged from 0.5 to 2.2-fold and no single conformation provided the best estimates for the genomes tested. Aside from the conformation of the DNA target gene, several variables between the three studies could account in part for the differences in magnitude of gene estimates observed between eukaryotic versus prokaryotic systems. Those include but are not limited to: 1) the conformation of the circular standard tested and 2) the preparation of the standards. In the Hou et al. study [7], the implication that the circular plasmid was supercoiled must be inferred from the text, as a gel image was not included. In any case, estimates were much higher than those using the linear standard. In the maize study [8], 3-fold inflation in gene estimates was observed for the supercoiled versus the linearized standard. Interestingly, both supercoiled and nicked 24195657 circular plasmids were prepared, but only the supercoiled was investigated for its affect on estimates using genomic DNA [8]. In results presented here, the effect of both circular plasmid conformations were assayed using microbial genomic DNA. Another source of variation was the method of standard preparation and quantification. Hou et al. purified the plasmids and amplicons prior to quantification based on the optical absorbance at OD260 [7]. Lin et al. demonstrated that supercoiled and linearized DNA showed differences in quantification based on the optical absorbance, but took measurements prior to purification [8]. In the present study, the digested plasmid standards and amplicons were purified andquantified following digestion or amplification to rid of enzymes or other contaminants that could interfere with quantification readings or com.