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RNA Isolation and Analysis:  First, we obtained samples of wild type yeast and zms1/zms2 knockout yeast.  These yeast cultures were grown in SD media and gently agitated overnight at 30ºC.  The yeast cells were collected in mid-log growth phase as shown by the nanodrop readings of 0.624OD (WT) and 0.600OD (double knockout) to prevent any age related variables in our data.  From here, yeast cells were broken open and RNA was collected according above protocol.  The isolation of the RNA was done with a RNAsafe kit from Qbiogene.  A nanodrop was then used to quantify the amount of RNA present in our samples.  Finally, the RNA was run on a gel electrophoresis to check for degradation.  These RNA's were used to make cDNA probes.  It is important to note that only 100μL of solution 3 was used when isolating RNA. 

Probe Construction:  Creating a labeled probe was accomplished using the Genisphere 3DNA Array 350 Detection Kit.  mRNA was reverse transcribed to cDNA which is done with a special primer with a capture sequence for red or green dye.  Red dye (Cy 5) was used for the double knockout cDNA, while green dye (Cy 3) was used for the wild type cDNA.  Next, the cDNA was concentrated into a much smaller volume using YM30 micro concentrators.  These probes were then used in the hybridization protocol.  Additionally, in step 9 of this protocol, the sample was incubated for 13 minutes at 65°C instead of 10 minutes. 

Hybridization:  Over a 48 hours, the hybridization of the cDNAs to the oligomer on the microarray slide (13760721).  The slides used for analysis are shown in table 1 with their corresponding dye labeling.  Grids 5 and 6 were analyzed from slides 13760694,13760722, and 13760724.  In addition to these grids, grids 21 and 22 were analyzed from slide 13760722.  This involved several steps including readying the slide, mixing the dyes with the probes, and washing the slide.  On the second day, under dark conditions the dyes with cDNA probes were added to slide and lefts to hybridize for 24 hours.  The following day, the slide was washed and shipped for reading.

Table 1:  Slide numbers and associated dye usage.  Slides 13760694, 13760722, and 13760724 were used for analysis.

The microarray slide was shipped overnight to Davidson College in NC for reading.  The results were sent back to our lab and read with Scanalyze and MAGIC TOOL to gain the results.  Magic Tool functions were used such as converting our values to log base 2 values and subsequently normalizing those values.  This was done to eliminate errors from background, dye bias, and other systematic errors.  Additionally, the mean normalization was set to 0.