Investigating Potential Changes in Gene Expression for Yeast Cells Exposed to Pediatric Flu Vaccine with and without Thimerosal

Investigating Potential Changes in Gene Expression for Yeast Cells Exposed to Pediatric Flu Vaccine with and without Thimerosal

As a control, gene expression in Saccharomyces cerevisiae (Baker's yeast) grown in standard YEPD media (normal growth conditions) was assessed using microarray technology and reverse transcriptase polymerase chain reaction (RT-PCR). Subsequently, yeast were grown in a typical dose of pediatric flu vaccine both with and without thimerosal preservative as experimental treatments. RNA from cells in each environment was extracted using the Ambion RiboPure Yeast Kit. RNA quality and quantity was assessed using gel electrophoresis, and UV spectrophotometry. The mRNA within the sample (about 1% of total RNA) was isolated by creating complementary DNA (cDNA). The presence and relative quantity of four genes of interest (SSA2, ECM4, RASI and SUA7) and a housekeeping (control) gene (TDHI) were tested for in the cDNA by using specific primers and PCR to amplify them. These segments were then visualized by gel electrophoresis. The genes of interest were selected for their particular cellular function and possible sensitivity to thimerosal. SSA2 is a chaperone — a molecule that assists in folding proteins. ECM4 is involved in glutathione (an antioxidant) transferase activity as part of a metabolic process that breaks down harmful free radicals and chelates heavy metals. RASI has an unknown function in yeast, but is believed to contribute to cancer risk in humans by playing a role in cell cycle control. Finally, SUA7 is involved in transcription (making RNA from DNA). T DHI is involved in metabolism and should always be expressed in functional yeast cells. Subsequently, cDNA from both vaccine treatments were fluorescently labeled and hybridized to yeast microarrays to test for global gene expression over the entire yeast genome (approximately 6,000 genes). Results of RT-PCR and microarrays were compared among all three treatments.