GeNorm analysis of candidate reference genes. (a) Ranking of candidate reference genes according to average expression stability. The least stable genes with the highest stability measure, M were excluded in a stepwise manner until the two most stable genes remained: miR-16 and miR-345. (b) Determination of optimal number of reference genes for normalisation. The GeNorm programme calculates a normalisation factor (NF) which is used to determine the optimal number of reference genes required for accurate normalisation. This factor is calculated using the variable V as the pairwise variation (Vn/Vn + 1) between two sequential NFs (NFn and NFn + 1). The number of reference genes is deemed optimal when the V value achieves the lowest, at which point it is unnecessary to include additional genes in the normalisation strategy. In this instance, the GeNorm output file indicated that optimal normalisation of gene expression could be achieved using the top five most stable reference genes.