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Upregulation of B-cell CLL/lymphoma (BCL)2 expression following lithium treatment is seemingly well established and has been related to the neuroprotective property of the drug. However, while demonstrated by some (but not all) studies based on low-throughput techniques (e.g. qPCR) this effect is not reflected in high-throughput studies, such as microarrays and RNAseq. This manuscript presents a systematic review of currently available reports of lithium’s effect on BCL2 expression. To our surprise, we found that the majority of the literature does not support the effect of lithium on  BCL 2 transcript or protein levels. Moreover, among the positive reports, several used therapeutically irrelevant lithium doses while others lack statistical power. We also noticed that numerous low-throughput studies normalized the signal using genes/proteins affected by lithium, imposing possible bias. Using wet bench experiments and reanalysis of publicly available microarray data, here we show that the reference gene chosen for normalization critically impacts the outcome of qPCR analyses of lithium’s effect on  BCL2  expression. Our findings suggest that experimental results might be severely affected by the choice of normalizing genes, and emphasize the need to re-evaluate stability of these genes in the context of the specific experimental conditions.

The observed alteration in BCL2 expression following lithium treatment is influenced by the choice of normalization method