Statistical analyses Sample size In general, the more biological replicates (larger sample size), the stronger the inferences that can be made from the gene expression data. Biological variation between samples and the expected spread of differential gene expression are important factors influencing the ideal sample size. For this study, the investigators do not know in advance how the gene expression levels would be distributed, thus restricting our ability to use sample size algorithms for RNA-seq experiments. In addition to the restrictions on sample availability discussed above, the high costs associated with laboratory processing limited the feasibility of using large numbers of AF samples.