However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression.
The use of this masking threshold significantly improved sensitivity (the number of expressed genes detected) while maintaining a high level of accuracy in measuring fold-difference in gene expression.
Next, we examined the effect of double-masking on the accuracy of measuring fold-differences in gene expression by plotted ΔΔCT values obtained from the real-time quantitative RT-PCR data against log2(252PES/1283PES) values from the GeneChip data before and after masking.
For example, if the RMS deviation is 0.15 (15%), then a measured fold change between samples of 1.15 is a difference of one standard deviation.
The ddCt method [ 35] was applied to measure relative fold differences between each strain and one wild type sample with act1+ used as the loading control.
The reliability index measures the fold difference between the highest scoring class and the second-highest scoring class.
To determine if the variability in measuring two-fold differences is independent of the problems associated with the choice of calibrators, we examined ΔCt values of offspring of a heterozygous line.
The effect size, measured in "fold-difference" between the means at which power was estimated at 0.80 was calculated for minor allele frequencies from 0.10 to 0.50 and alpha levels of 0.05 and 0.00021 (the Bonferroni correction for 233 tests) assuming markers do not deviate from Hardy-Weinberg Equilibrium (table 2).
A statistical power analysis indicated that three rabbits are theoretically required to provide sufficient power for this study, and this was also true when accounting for several fold differences we measured in our assays http://www.dssresearch.com/toolkit/sscalc/size.asp.asp
Two polymerase (RNA) II polypeptide genes, POLR2B and POLR3A, showed higher expression in the SCNT embryos with an average of 6.3- and 5.2- fold differences, respectively, when measured with qPCR and 2.8- and 3.2-fold differences, respectively, using microarray measure.
