Validation of EAR as a tool for normalization in RT-qPCR. (a) Application of the GeNorm algorithm to eight genes to identify the three most stable housekeeping genes in blood. Average expression stability values (M) of remaining control genes during stepwise exclusion of the least stable control gene in the human blood of eight control participants. (b) Blood BDNF mRNA relative to EAR and to the three most stably expressed housekeeping genes. Pearson's correlation r = 0.56; P < 0.05. (c) Median blood BDNF mRNA levels versus GAPDH, versus the three most stable control genes and versus EAR in ten control participants. Samples used in (a-c) were from the National Neurological Institute-IRCCS "Carlo Besta", Milan (Italy). (d) Median blood BDNF mRNA levels versus GAPDH, versus β2M, and versus EAR mRNA in eight control participants from the Institute of Neurology, MRC Prion Unit, University College London (UK).