Materials and Methods

Results

Discussion

Literature Cited

Introduction

Title Page

    Wild-type and mutant (Δzms1, Δzms2 and Δzms1/Δzms2) yeast cultures were grown in SD media to late log-phase, approximately 1.5-2.5 optical density at 600nm. At this point, the cell walls were enzymatically degraded to form spheroplasts, which are more easily lysed. RNA isolation (RNA Isolation Protocol) was performed using the RNAsafe kit from Qbiogene. Following isolation, a nanodrop was used to determine concentration and purity of the RNA samples. Also, agarose gel electrophoresis was performed to check for RNA degradation.

     RNA samples were labeled for microarray analysis (Labeling Protocol) using the Genisphere 3DNA Array 350 Detection Kit. Each sample of mRNA will be reverse transcribed into cDNA using a poly-T primer with a 3DNA capture sequence for specific dye (either Cy3 or Cy5). Our wild-type cDNA was labeled with Cy3 (green) dye primer and Δzms1 cDNA was labeled with Cy5 (red) dye primer. The cDNA was then concentrated using YM30 micro-concentrators. We obtained 11μL of concentrated cDNA.   

     The microarray slide (# 13760722) containing 70mer oligos and 2 copies of all known cDNAs in the yeast genome were pre-hybridized with a blocking buffer. The slide was then hybridized with the labeled cDNAs (2-Step Hybridization of DNA Chip Protocol). After washes of increasing stringency to remove unbound cDNA, the dyes were put onto the slide in a second hybridization. After washing the slide again, with washes of increasing stringency, the slide was packaged in liquid nitrogen and shipped to Davidson College for scanning.  

    The data from grids 7 and 8 on each of the microarray slides listed in Table 1 were analyzed using the programs ScanAlyze and MagicTool. 

 Table 1. Microarray slides used in statistical analysis of data to compare Δzms1 gene expression to Δzms1/Δzms2 gene expression.