( (for determining C(t)) was set manually...)Monitoring Microbial Populations Using Real-Time qPCR on the MJ Research Opticon 2 System

(for determining C(t)) was set manually to 0.015 fluorescence units for all experiments, using the Opticon Monitor software (MJ Research). This threshold is automatically applied to all wells for consistent analysis of individual samples and standards.

Quantification Analysis
All samples were analyzed in triplicate. The cycle threshold (C(t)) values for amoA were determined for each sample and for the control plasmid- DNA template. The amoA target was normalized to the plasmid-DNA control using the following calculation: ΔC(t)_sample = average-C(t)_amoA average-C(t)_plasmid-DNA. The normalized sample C(t) values were then referenced to one soil sample (the calibrator) to determine the relative effect of the environmental treatments on AOB levels. The formula used for determining relative amoA levels is as follows: ΔΔC(t)_sample = ΔC(t)_sample ΔC(t)_calibrator. The amount of amoA for each soil sample is reported as a ratio relative to the calibrator using the following formula: 2^ΔΔC(t) (ref. 9).

Results
Relative Quantification of amoA Levels for Soil Plots
Each of the 16 samples presented in this report was subjected to a different combination of environmental factors: temperature, CO₂, precipitation, and nitrogen deposition. Table 3 shows the mean C(t) standard deviation as well as successive calculations for relative quantification of the amoA gene in four representative samples. Note that sample 1 has been designated as the calibrator to which the other three samples were normalized. The standard deviation for triplicate measures of each sa
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