Appropriate Reference Gene for the Gene Expression Analysis in U87 Glioblastoma Cell Line

Cancer is one of the most challenging diseases in the world. It is widely accepted that knowing the molecular aspects of diseases, including cancers, helps to develop methods for their therapy and diagnosis. Long non-coding RNAs (lncRANs) are a novel category of regulatory genes known to be involved in cancer incidence. The expression of these genes is said to be suitable of using in prognosis, diagnosis, targeted therapy, etc. The RT-qPCR method that is widely used for analyzing the gene expression requires the application of appropriate reference genes as the internal control. The expression status of a proper housekeeping reference gene is not supposed to change under experimental circumstances. This study aimed to find a suitable reference gene in the U87 cells after overexpression of a gene of interest. To this aim, the expression status of four common reference genes (ACTB, β2M, GAPDH, and HPRT1) was examined in the transfected U87 cells. The U87 cells were transfected with a vector overexpressing YWHAE-lncRNA and an empty vector (mock). After total RNA extraction and cDNA synthesis, RT-qPCR was applied using the aforementioned internal control genes. Data were analyzed, and their graphs were plotted in GraphPad Prism 8.2 software. Β2M showed the most change; accordingly, GAPDH and HPRT1 expression levels were changed about 5 and 4 times, respectively. Of the candidate genes, only the ACTB gene had a consistent expression level in two different modes of transfection, and therefore, it is suggested as an appropriate reference gene for the study of gene expression in the transfected U87 cell line. It is remained to be tested if β2M, GAPDH, and HPRT1 common internal controls are specifically affected by YWHAE-lncRNA overexpression or other lncRNAs may affect their expression as well.