TRANSCRIPTIONAL CHANGES OF MYCOPLASMA CONTAMINATION IN GENE EXPRESSION STUDIES

Abstract

Mycoplasma, the smallest self-replicating microorganism, is one of the most significant problems in the cell-culture field. It characterized by the absence of the cell wall, causing resistance to commonly used antibiotics in cell culture. The frequency of mycoplasma contamination has reached up to 35% worldwide, while the incidence found to be higher (42%) in Qatar. Numerous gene-expression studies have shown that mycoplasma dis-regulates thousands of genes on cultured cells causing unsafe biological products and erroneous experimental results. Therefore, it is critical to ensure that transcriptional changes due to mycoplasma are eliminated and gene-expression profile is restored to normal level after anti-mycoplasma treatment, to avoid biased results. For this purpose, first, we developed a survey to investigate the incidence of mycoplasma contamination in cell-cultures and most common methods used for mycoplasma detection and elimination in research laboratories in Qatar. Second, we used next-generation sequencing technology (illumina 4000 Hi-seq) to investigate the effect of mycoplasma on gene-expression profile of cultured cells and the ability of antibiotic treatment in reversing these changes. Results showed that number of dis-regulated genes in contaminated cells is increasing with the increase of sub-culturing. Affected genes were mainly related to cell death and survival and cell-to-cell signaling and interaction. Interestingly, genes dis-regulation was not only due to mycoplasma contamination, antibiotic treatment also contributed to the effect. Unlike the effect of mycoplasma, antibiotic treatment showed a shrinking pattern on the number of dis-regulated genes. Findings of this study concludes that mycoplasma contaminated-cells should not be further used or processed, specially for gene-expressions studies, due to the significantly dis-regulated genes as consequence of either mycoplasma or antibiotics effects.