Ranking of candidate reference genes in order of stability by geNorm. (A) Expression stability values (M) of candidate reference genes in 5 h and 24 h collagenase-induced ICH and saline vehicle rats. geNorm ranks reference genes in order of least stable to most stable (left to right) by stepwise exclusion of the least stable gene. (B) Pairwise variation (V) analysis determines the optimal number of reference genes that should be used for accurate normalisation, with a threshold value of 0.15 . In this case, the V3/4 step achieves the cut-off value, and therefore, the appropriate number of reference genes for accurate normalisation is four. The far right bar represents pairwise variation when the three most stable genes are removed from analysis and the stability of the remaining genes (GAPDH, HPRT, SDHA and TBP) are re-assessed. In this case the V threshold is not met.