Materials
Wild type and ∆ZMS1 mutant yeast cell lines were courtesy of Dr. Kim Slekar of James Madison University
Microarray kit used was purchased from Genisphere
Microarray slides purchased from GCAT
Microarrays were scanned at Davidson College
Methods
RNA was isolated from yeast cells (Saccharomyces cerevisiae) that were in the mid log-phase according to the protocol found here. In step 8 of the section "preparing yeast cells by making spheroplasts," the supernatant must be decanted prior to repeating step 7 as stated. Also in step 9, no sterile water was needed. Then, we ran 2.0µg of RNA on a 1.2% agarose gel as stated to check for RNA degradation.
Next, we had to convert the isolated RNA to more stable cDNA for use on a microarray. The conversion and labeling procedures can be found here. In step 2 of the "reverse transcription" protocol, we used 2.0µg of previously isolated yeast RNA suspended in 12µL of DEPC treated (nuclease-free) water mixed with 1.0µL of the appropriate primer (Cy3 for wild type and Cy5 for the mutant). Step 6 of the protocol was also altered so that we used a solution composed of 4.0µL 5X RT buffer, 1.0µL 10µM dNTPs, 1.0µL Superase-In and 1.0µL reverse transcriptase. Then in step 7, we only added 7µL of the above solution to the suspended RNA. 6.0µL of cDNA were recovered.
All of the cDNA generated from the isolated RNA was then hybridized onto a microarray using this procedure. During the pre-hybridization the slide was placed in a 55°C incubator (step 5). In step 2 of the first hybridization, 6µL of the concentrated cDNA and 21µL of nuclease-free water were added to the other components for a total volume of 58µL. During the final washing procedure, an error occurred in step 13 which resulted in the slide being placed in 0.2SSC 1mM DTT for approximately 1.5 minutes. When the error was realized, the slide was then transferred into the correct solution and for steps 14 and 15, remained in the relevant solutions for only 10 minutes in an attempt to mitigate the effects of the initial error.
Normalization
Due to extensive photobleaching of the Cy5 dye, data from two different microarray slides were used. The slide for which Cy3-labeled wild-type cDNA was used was #13452127 and the slide for which Cy3-labeled mutant cDNA was #13452129. The program ScanAlyze was used to visualize, grid and quantify signal intensities for the two slides used.
The first step of normalization was comparing the signal intensity of the green-mutant signal to the green-wild type signal (Mutant Signal / Wild type Signal). This ratio was then transformed into a log2 value. All computations were performed using Microsoft Excel®. At this point the signal ratio of the housekeeping genes was averaged, and every gene signal was compared to this average. Any signal demonstrating a four fold induction or reduction was deemed of interest. However, hundreds of genes were "of interest" using this methodology, so a new method was used. Normalization was ultimately performed by averaging all of the log2 signal ratios for the empty (negative control) wells and the randomly generated negative controls (spots containing randomly generated DNA with a similar G/C content as yeast genes). A gene of interest was then defined as any ratio that exhibited a three standard deviation change from the averaged negative controls. Three standard deviations were chosen because the general consensus in the scientific community is that two standard deviations indicated a change assuming multiple trials have been performed. In this experiment only one trial was performed; therefore in order to compensate three standard deviations from the mean were used.
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