Materials & Methods
Yeast Culture Preparation:
Yeast cultures were prepared for RNA extraction according to the following protocol
Day 1:
Yeast cells were extracted from a plate culture using sterile popsicle stick and placed into liquid media. They were then incubated in a shaker at 30°C overnight.
Day 2 (very early morning~6:00 AM):
Spectrophotometer readings were taken of each liquid culture at 600nm. These readings were used to determine the dilution factor needed to obtain 90mL of fresh yeast cultures at an OD of 0.3. The 90mL cultures of yeast at an OD of 0.3 were incubated in the shaker at 30°C for approximately 7.5 hours.
RNA Extraction:
RNA was extracted from yeast in the mid-log phase of growth. Yeast cells were turned into spheroblasts using b-mercaptoethanol and lyticase in preparation for RNA extraction. Glass beads were used to lyse spheroblasts and RNA was extracted using the RNAsafe kit (Qbiogene). The concentrations and purity of extracted RNA was determined using a spectrophotometer. Portions of the extracted RNA was run on a 1.2% agarose gel to check for quality.
Link to complete protocol.
All precautions were taken to prevent contamination of samples with RNases.
cDNA Probe Construction:
A poly-T primer with a capture sequence for either the Cy3(green) or Cy5 (red) were used to prepare cDNA probes. The Cy3 capture sequence was used for control wildtype yeast and the C5 capture sequence was used for ZMS2++ yeast. Primers were incubated the extracted RNA and reverse transcribed in order to obtain the cDNA probes. These cDNA probes were concentrated using YM30 microconcentrators.
The RNA concentrations that were obtained from our extractions were too low to make a good amount of cDNA. RNA from other groups were used to make out cDNA.
Problem encountered during protocol:
During the final spin of the extracted RNA (step 8 in the protocol), the microcentrifuge broke up parts of the microcentrifuge tubes’ tops and had to be stopped immediately. The cycle only ran for a few seconds and was not allowed to finish.
Link to complete protocol.
Hybridization:
Slide #13697524
DNA microarray chips were prepared with steam and pre-hybridized using a solution of 3x SSC. 0.1% SDS, and 0.1mg/mL sonicated salmon DNA. Chips were hybridized with cDNA at 45°C for at least 24hours. Following hybridization with cDNA, chips were washed and hybridized with the Cy3 and Cy5 dyes with an anti-fade reagent and incubated overnight at 55°C. The following morning, slides were washed and sent to Davidson College in a N2 atmosphere.
The green dye was for the wild type control yeast while the red dye was used for the ZMS2++ strain.
Link to complete protocol.
Data Analysis:
Slide used for analysis: Slide # 104 from 2004, a ΔZMS1ΔZMS2 double knockout
The data was gridded using Scanalyze and converted to an excel file. The intensity of the background of the green dye was subtracted from the intensity of the green dye in each spot and the same was done for the red dye. For both dyes, any spot where the difference between the intensity and the background was zero or a negative number was deleted. The intensity of the red spots was then divided by the intensity of the green spots. Any number that was divided by zero was deleted. These values were then converted to logarithms base 2.
Common Promotor Regions for Upregulated Genes Protocol:
The average and standard deviation of log to the base 2 of the red/green ratio of eight microarray slides was calculated. The data included slides 104 and 106 from the Fall 2004 class data. The most extreme average -2.71 with a standard deviation of 1.62 was used as the parameter for over expressed and under expressed genes.
Using the explore function on Magic Tool, four of the 106 slides were analyzed to find over expressed genes. Four genes that were over expressed, with a log base 2 ratio greater than -1.09 in all four slides were found. The function of the gene as well as the sequences of the promoter regions were found using the Saccharomyces Genome Database. The transcription factor binding sites were found by running the sequences on the YeastTract website. Transcription factors that were common across all four genes were found.
Over expressed and Under expressed Genes in ZMS1DZMS2D Protocol:
MagicTool and Excel were utilized in finding the most over expressed and under expressed genes in the ZMS1DZMS2D slide (slide number 106). The average intensity of each slide were calculated using the log2 ratio values of each gene. All of the averages were computated using Excel and the standard deviation of intensity for each slide was calculated. The most extreme average plus or minus the standard deviation was used as the parameter for intensities of over expressed and under expressed genes. The most extreme average was -2.71 with a standard deviation of 1.62.
The expression file of the bottom of the ZMS1DZMS2D slide was analyzed using the explore function of MagicTool. The average intensity + 3 standard deviations was used as the expression value for over expressed genes. The average intensity - two standard deviations was used as the expression value for under expressed genes. The number of standard deviations used for over expression and under expression was determined by the number of genes given that matched the criteria. More than one standard deviation from the average had to be used in order to narrow the explore results to only those with the greatest and smallest expression values. After the bottom portion of the slide was analyzed, the corresponding top portion of the slide was also analyzed using the explore function. A similar protocol was used to determine the most over expressed and under expressed genes.
In order to determine if the genes over expressed and under expressed in the slide were similar, the expression files of the top and bottom were loaded as a single file into MagicTool. The explore function was utilized to find genes which were over expressed and under expressed in both the top and bottom of the ZMS1DZMS2D slide. A parameter value of the average intensity + two standard deviations was used for over expressed genes and a value of the average - one standard deviation was used for under expressed genes.
A comparison of the over and under expressed genes in the ZMS1DZMS2D slide and ZMS1D slide was made to determine if any of the same genes were commonly over expressed or under expressed. A similar protocol was used to determine the over expressed and under expressed genes in the zsm1D slide. The expression file of the top of zsm1D slide (slide number 104) was loaded into MagicTool. The explore function was utilized to determine how the genes over expressed and under expressed in the ZMS1DZMS2D were expressed in the ZMS1D slide. The expression value of each gene on the ZMS1D slide was found, added to an Excel file and then compared to the expression value of the ZMS1DZMS2D slide. The same parameters were used for over expressed and under expressed genes in the ZMS1D slide as for the ZMS1DZMS2D slide.
The function of each gene was found using the GeneList data for the 2004/2005 Yeast Chips.