Materials and Methods
The following protocol
can be accessed on the James Madison University Biology 480/580 website. Hyperlinks are provided as direct links to
specific pages at the end of each protocol.
Culture Preparation
Yeast cultures in 50 ml quantities were grown in SD media the
night preceding the experiment. Approximately six hours prior to RNA
harvesting, cultures were restarted in SD media to maximum yeast cells growing
in log phase. Immediately prior to RNA harvesting the optical densities
(600 nm) were taken to ensure equal numbers of yeast cells in all samples.
RNA Isolation
The enzyme
lyticase was used to break down the yeast's outer cell wall. RNA
isolation was preformed using the RNAsafe kit (Qbiogene). This kit
contained several agents meant to reduce the action of RNAse, the enzyme
responsible for RNA degradation. Special care was taken not to introduce
any RNAses to the samples throughout RNA isolation.
Cells were lysed by 10 intervals of 30 second vortex and 30 second on ice. The ice served both to reduce enzyme activity and to help shatter cellular membranes. The samples were centrifuged and the supernatant harvested to separate out the membranes, unbroken cells, and proteins. DNAse was added to get rid of the DNA in our samples. The amount of RNA and the purity was checked using a Nanodrop spectrophotometer. A electrophoresis gel was also run to check for RNA degradation and the presence of DNA.
cDNA Probe Construction
Probe Construction
Using our RNA samples (2.4 ug WT, 6.4 ug ZMS1) and the Genisphere 3DNA Array 350 Detection Kit cDNA probes were created. The mRNA samples were reverse transcribed for 1 hour using primers including a poly-T tail that will bind to the poly-A tail of mRNA and a specific 3DNA capture sequence that will bind to our dyes. Our WT cDNA was set up with the green dye Cy3 and our ZMS2delta cDNA was set up with the red dye Cy5.
Concentration
After combining our cDNA samples into a single tube, a YM30 Microconcentrator was used along with high centrifugation to separate out nucleotides and other micromolecules from our sample. Our concentrated samples were stored at -70°C until use.
Hybridization (slide
113)
Yeast DNA microarray chips containing two copies of the 70mer oligonucleotides that represent the known yeast genome were washed with salmon sperm DNA to neutralize the positive charge covering the slide. The cDNA was hybridized to the slide (number 113) followed by a wash to remove any unbound cDNA. Then the dyes were hybridized to the slide, binding specifically to the cDNA with the special capture sequence. After a second wash to remove the unbound dyes the slides were shipped to Dr. Campbell (Davidson College, North Carolina) for scanning.
Anaylisis Of Microarray Data
We were unable to get any useful data from our slides. Further anaylisis was done using slide 104 data from previous micrroarray experiments (2004). The slide 104 image was gridded and processed using ScanAlyze software. Data from ScanAlyze of all four groups were statistically analyzed in MagicTool software to highlight interesting gene expression patterns. Information about individual genes was gathered from the GCAT website. Upstream gene sequences were collected from the yeast genome. The down regulated, genes not readily expressed, collected from magic tool were compared to try and identify if there were any conserved sequences present with Megaline.