Real-time PCR assay for the quantification of RNA transcripts involved in the control of globin gene switching

Abstract

In this project, the globin gene switch was studied using an innovative combination of small molecule screening and a transcriptomic approach using real time qPCR. This allowed the exciting possibility of directly correlate and assessing the role of small molecules in HbF induction. An in vitro cell culture model system was developed using K562 cell lines and a real time qPCR method optimized on globin transcripts. Induction of γ-globin reduces symptoms in β-thalassaemia and sickle cell disease patients but there is a clear need for drugs with a better efficacy, tolerability and patient response. At present, treatment involves lifelong monthly blood transfusions and iron chelation therapy, both of which impinge significantly on the quality of life and may lead to death prematurely. To the K562 cell line, three novel compounds provided by the European Molecular Biology Laboratory, Heidelberg were added. The correct cell number and drug concentrations were calculated after conducting cell viability assays, and subsequently ribonucleic acid was extracted from this cell line for quantification by real-time PCR to see whether these drugs have an effect on both proliferating and haemin-induced differentiating K562 cells. It was concluded that the drugs do have an effect on the K562 cells in vitro. This study can be beneficial for the therapeutic targeting in the treatment of haemoglobinopathies. Uncovering the molecular basis of γ-globin gene control might serve as a model for other clinically significant gene switch mechanisms.