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RNA Isolation and Analysis 

Preparation:   

    RNA had to be isolated from the Sacromyces cerevisiae in order to be analyzed.  Wild type and ∆ Hexose strains of Sacromyces cerevisiae were cultured by instructor to obtain optimal concentrations.  The absorbance of both samples were taken at a wavelength of 600nm to determine what growth phase the yeast were in.  The cell walls of the yeast were broken down with beta-mercaptoethanol to hydrolyze the disulphide bonds and lyticase.  The created Spheroblasts can easily be ruptured to obtain the RNA from the cytosol.

Isolation:

    The isolation was preformed using the RNAsafe kit from Q-biogene.  The cells were lysed and detergent solutions were used to inactive cellular RNAses.  Special care was taken to avoid RNAase contamination.  The RNA was placed in a quartz cuvette and quantified by measuring the absorbance at 260nm with a spectophotometer.  Electrophoresis was applied to run the RNA on an 1.2% agrose gel.  

Probe Construction

    The probes were constructed with the Genisphere 3DNA array 350 detection kit.  Again special care was taken to avoid RNAse contamination.  The wild type probe was labeled with green dye and the mutant probe was labeled with red dye.  The RNA was then reverse transcribed to form cDNA. Th cDNA was concentrated so that it would take up as lesser volume. 

Hybridization

    First the micro array slide had to be treated to remove excess salt and to denature any folding that might have previously occurred between the oligos.  The slide was then prehybridized with sonicated herring sperm DNA.  The cDNA was hybridized to the oligos and incubated until the following afternoon.  Twenty Four hours later, a wash was preformed and the dye was hybridized to slide.  During this procedure the lights were turned off to avoid exposing the dyes and incubated until the next morning. A final wash was preformed and then the micro arrays were shipped to Davidson College for scanning.            

Analysis of data on Magic Tool:

Slide numbers used: TR3302 w595 (green, wild type) and TR3302 w685 (red, delta hexose mutant) from fall 2004

The slide image of the microarray was gridded and segmented using the program Magic Tool. Then the ratios of red to green were computed for each gene spot on the slide. The dissimilarities between each expression ratio were calculated, and this data was displayed using hierarchical clustering.

Refer to: http://www.bio.davidson.edu/projects/magic/magic.html      for more information on how Magic Tool works.