Materials and Methods
Step One - RNA Isolation and Analysis
Full Procedure: http://csm.jmu.edu/biology/courses/bio480_580/mblab/RNA.htm
Prep:
Yeast cultures were grown SD media on Day 0. Day 1 of the experiment, the samples were started. The samples were analyzed at 600nm to compare the number of yeast cells in each culture. Two cultures were cultivated: a ZMS2 mutant and a wildtype culture.
RNA Isolation:
Initially, the cells were made into spheroplasts by use of the enzyme lyticase. Following this, the cells were broken. Using an RNAsafe kit, the RNA was collected and kept on ice. The RNA absorbance was measured at 260nm by use of a nanodrop.* The Absorbance at 280 was taken to examine the purity of the RNA. A sample of the RNA was then run on an agarose gel with ethidium bromide as a dye. The RNA was examined prior to Step 2 in order to determine a good sample with which to continue.
Step Two - Probe Construction
Full Procedure: http://csm.jmu.edu/biology/courses/bio480_580/mblab/label2.html
For this step, cDNA microarray probes were created. A separate probe was created for wt and ZMS2 mutant.. The probes were labeled with a Genisphere 3DNA Array 350 detection kit. Initially, the RNA was mixed with the primer tags, Cy3 (green) for the wt and Cy5 (red) for the mutant. The mixture was heated next, to remove any secondary structure. Reverse Transcriptase was added to the solution, along with buffer and various dNTPs. The solution was then incubated for an hour and a half. The reaction was stopped with the addition of EDTA and the solution was incubated for another 10 minutes, denaturing the RNA and leaving only DNA.
Finally, the DNA was concentrated by use of a YM30 microconcentrator, and left at -70 C for one week.
Step Three - Hybridization
Full Procedure: http://csm.jmu.edu/biology/courses/bio480_580/mblab/dnachip2.htm
To yeast microarray chips were washed with salmon sperm DNA to neutralize the charge on the slide. The cDNA was hybridized to the slide, and washed to remove any unbound cDNA. The dyes were hybridized to the cDNA, thus labeling the DNA attached to the slide. The slide was washed again, and shipped to Davidson College, North Carolina for analysis. In process, the slide containing the sample was broken. Further observations will be conducted with a previously made slide.
Step Four - Analysis
The results were analyzed utilizing Microsoft Excel and Magictool. Due to the loss of our microarray slides, slides from a previous year were used. This slides was 726 from Fall 2008 ZMS2 labeled with red. The data was compared and upregulated along with downregulated genes were noted.
* Denotes change in original procedure.
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