Evaluation of Reliable Reference Genes for In Vitro Erythrocyte Generation from Cord Blood CD34 + Cells

Abstract

In vitro generation of red blood cells has the potential to circumvent shortfalls in the global demand for blood for transfusion applications. However, cell differentiation and proliferation are often regulated by precise changes in gene expression, but the underlying mechanisms and molecular changes remain unclear. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) can be used to evaluate multiple target genes. To make the results more reliable, suitable reference genes should be used to calibrate the error associated with qRT-PCR. In this study, we utilized bioinformatics to screen 3 novel candidate reference genes (calcium and integrin binding family member 2 [CIB2], olfactory receptor family 8 subfamily B member 8 [OR8B8], and zinc finger protein 425 [ZNF425]) along with eight traditional reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], β-actin [ACTB], 18S RNA, β2-microglobulin [β2-MG], peptidylprolyl isomerase A [PPIA], TATA box-binding protein [TBP], hydroxymethylbilane synthase [HMBS], and hypoxanthine phosphoribosyltransferase 1 [HPRT1]). Two software algorithms (geNorm and NormFinder) were used to evaluate the stability of expression of the 11 genes at different stages of erythrocyte development. Comprehensive analysis showed that expression of GAPDH and TBP was the most stable, whereas ZNF425 and OR8B8 were the least suitable candidate genes. These results suggest that appropriate reference genes should be selected before performing gene expression analysis during erythroid differentiation and that GAPDH and TBP are suitable reference genes for gene expression studies on erythropoiesis.