oa Transcriptional Changes of Mycoplasma Contamination in Gene Expression Studies

Mycoplasma, the smallest self-replicating microorganism, is one of the most significant problems in the cell-culture field. It belongs to the class of Mollicutes, which are characterized by the absence of a cell wall, making them resistant to commonly used antibiotics, such as streptomycin and penicillin. Because of their small size, Mycoplasmas can pass through the 220-nm pores of filters used to sterilize culture media. Owing to these criteria, contaminated cell-culture with Mycoplasma not easily detected for extended periods of time, since Mycoplasma cannot be visualized by the naked eye or even by light microscopy and do not produce overt turbid growth commonly associated with bacterial and fungal contamination. The three major sources of Mycoplasma contamination in cell-culture are 1) Cross-contamination from infected cultures, 2) contaminated culture reagents (e.g. serum and trypsin), and 3) infected laboratory personnel with M. orale or M. fermentans. Knowing the importance of cell-culture as a significant research tool for a variety of biomedical disciplines, several effective methods and kits were developed to detect Mycoplasma contamination of cell-culture and others to eliminate the contamination using antibiotics.

The frequency of Mycoplasma contamination in cell-culture has been extensively discussed in the literature. Previous reports show high incidences of Mycoplasma contamination reaching up to 35% worldwide, with extreme incidences of 65% in Argentina and 80% in Japan.

Mycoplasma contamination of cell-culture has diverse and comprehensive consequences, ranging from unsafe biological products, to erroneous experimental results. Due to the lack of amino acid biosynthesis genes, Mycoplasma competes with its' host cell nutrients and biosynthetic precursors, leading to DNA, RNA and protein synthesis alterations. Using microarray platforms, numerous gene-expression studies have shown that Mycoplasma contamination significantly up regulates and down regulates thousands of genes like oncogenes, tumor suppressors, cytokines and growth factors. Therefore, in gene-expression studies, it is critical to ensure that transcriptional changes due to Mycoplasma contamination in cell-culture 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 the techniques frequently used for the detection and elimination of Mycoplasma in research laboratories at Qatar. Based on the output of this survey, SKOV3 ovarian carcinoma cells, LookOut® Mycoplasma PCR Detection Kit from Sigma and BM-Cyclin from Roche were selected as most commonly used cells, detection kit and elimination kit of Mycoplasma, respectively.

Second, we used RNA-seq technology, for the first time, to investigate the transcriptional changes of Mycoplasma contamination in cell-culture and the ability of anti-Mycoplasma treatment in reversing these changes.

RNA samples extracted from uncontaminated (control), Mycoplasma-contaminated and anti-Mycoplasma treated cultured cell (Skov3) were sequenced on illumine 4000 Hi-seq. Generated data were analyzed by comparing gene expression profiles of these three conditions and summarizing all differentially expressed genes and their associated pathways. Preliminary data demonstrated that mycoplasmas alter the expression of hundreds of genes in cultured Skov3 cells. A variety of pathways are affected, and both up-regulation and down-regulation were seen. Deeper analysis specifying significantly altered pathways are in progress along with analyzing data of the post anti-Mycoplasma treatment.