Comparing these large numbers of genes with significant gene expression in at least one batch to the number of genes differentially expressed between sets of replicate samples for the two experiments (85) suggests that both within- and between-experiment batch effects are of a sizeable magnitude in this data set.
Pair-wise statistical analyses were performed using Student's t-tests between sets of biological replicates from different treatments collected during the same time period only.
Biological correlation of gene expression was examined between the Affymetrix and Nanostring platforms, and between the two sets of replicates processed on the Nanostring platform.
Scatter plots of the log2 expression measures between a set of replicates processed with different methods and Pearson correlation coefficients were prepared using Excel 2003 (Microsoft, Redmond, WA).
Principal component analysis (PCA) of 8 Affymetrix arrays showed high variation between the two sets of replicated arrays that were processed on two distinct days (day1 and day2).
Remarkably, the average absolute difference between the 'pooled' DNA methylation estimate and the 'real' average, determined by assessing individual samples, was 6.0% in the first set of experiments and 4.8% in the second set of replicates.
When we apply MDS to Robinson-Foulds distances between the sets of bootstrap replicate trees produced by pairs of strategies, we obtain similar observations of the clusters of strategies, detecting an important role for M and for the difference between topologically-based and non-topologically-based strategies, and no strong signal for the outgroup choice.
To further investigate the robustness of our RNA-Seq dataset, the correlation coefficients of the transcriptome profiles among the eight samples were calculated and were found to reach 0.99 between each set of biological replicates (Additional file 1: Table S3).
For the tissue sample displayed in Figure 1, the variability associated with technical replicates is larger for the 1.5 K panel in comparison to the 24K panel; the standard deviation associated with the difference in expression between the set of technical replicates shown is 0.551 for the 1.5K panel and 0.214 for the 24K panel.
This validation test included a set of replicate RNA from the microarrays plus six independent biological replicates.
