A main limitation of efficiency calibrated method and ΔΔCt method is that only one set of cDNA samples are employed to determine the amplification efficiency. It was assumed that the same amplification efficiency could be applied to other cDNA samples as long as the primers and amplification conditions are the same. However, amplification efficiency not only depends on the primer characteristics, but also varies among different cDNA samples. Using a standard curve for only one set of tested samples to derive the amplification efficiency might overlook the error introduced by sample differences. In our experimental design, we have performed standard curve experiments with four concentrations of three replicates for all samples and genes involved. The ΔΔCt will derive from the standard curves only, and the data quality is examined for each gene and sample combination. The analysis of two samples is presented in the paper as an example. A minimal of PCRs of two replicates in three concentrations will be required for each sample. Even though more effort is required, the data is more reliable out of stringent data quality control and data analysis based on statistical models.
The output dataset included Ct number, gene name, sample name, concentration and replicate. We used Microsoft® Excel to open the exported Ct file from an ABI 7000 sequence analysis system and then to transform data into a tab delimited text file for SAS processing. The sample data set is shown in Table 1.
All programs were developed with SAS 9.1 (SAS Institute).
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