Establishing a Control of Gene Expression in Yeast Exposed to a Standard Environment as a Comparison to Yeast Exposed to Flu Vaccine With and Without Thimerosal

Establishing a Control of Gene Expression in Yeast Exposed to a Standard Environment as a Comparison to Yeast Exposed to Flu Vaccine With and Without Thimerosal

Gene expression in Saccharomyces cerevisiae (Baker‘s yeast) was assessed using microarray technology. After growing the yeast in standard conditions (YEPD media), the total RNA was extracted using the Ambion RiboPure Yeast Kit. The quality and quantity of the RNA was assessed using gel electrophoresis and UV spectrophotometry. Next, mRNA (approximately 1% of the total RNA) was isolated using reverse transcription reactions. cDNA was made from the mRNA by amplifying a gene that should always be expressed (TDHI) using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Several key genes that are hypothesized to be affected by thimerosal in vaccines were also tested for in the sample of cDNA: SSA2, ECM4, RAS1 and SUA7. SSA2 is a chaperone that assists in folding proteins. ECM4 is involved in cell wall organization. RAS1 has an unknown function, but it is believed to contribute to cancer risk in humans. Finally, SUA7 is involved in transcription of DNA to RNA. The cDNA that was created from the mRNA was tagged with a fluorescent dye and hybridized to a microarray which was subsequently assessed using a program called MAGICTool. The resulting microarray image contained features (spots) that were clear and distinct from the background. After analyzing the microarray data, expression ratio values of the genes of interest were as expected and similar to the results obtained from RT-PCR reactions. Future applications of this procedure include exposing the yeast culture to vaccine with and without the preservative thimerosal and comparing gene expression between all three enviro nme nts.