In order to obtain a cDNA microarray side, RNA must first be isolated from yeast. The RNA Isolation and Analysis protocol was used. Mutant and wild type cultures were obtained by a previous growth experiment. The cell walls were enzymatically degraded. Using an RNase-free environment, RNA was obtained through a series of chemical treatments and centrifugation. RNA was then quantified and absorbance assay using a spectrophotometer at an absorbance of 260 nm. The concentration will be determined using the following equation:
O.D. reading x ((40 ug/ 1000 ul )/ 1 O.D. unit )x dilution factor = concentration in ug/ ul
The RNA was then checked for degradation using gel electrophoresis. Since the rRNA is the most abundant type, the strands making this up will be seen. It was expected that there be thick 28S, 18S, and 5S bands. The results were analyzed for quality using Nano-drop spectroscopy and gel electrophoresis according to the RNA Quality Guide. Nano-drop spectroscopy was then employed specifically to determine concentration and purity of RNA. The A260/A280 of quality RNA samples is above 1.8 meaning there is high purity.
To make the cDNA for microarray hybridization, the Probe Construction procedure was followed. First, the RNA obtained from the previous experiment was probed with a poly-t tail. The Genisphere 3DNA Array 350 Detection Kit was used to make the labeled probes. Reverse transcription was then performed on the RNA to produce cDNA which was then concentrated down to a smaller volume in order to be used for hybridization.
Hybridized to the microarray slide was performed by following the Hybridization procedure. The wild type RNA was dyed green while the mutant ZWSD2 was dyed red. A separate lab group performed the dye reversal.
The data was analyzed using Scanalyze software, which calculated the amount of pixels of light in each dot. The data was then transferred into excel where the background noise was subtracted and a value was obtained for mutant vs wild type. This data was inputted into Magic Tool v 2.1 where we did a log base 2 transformation and subsequent normalization and standardization. The data was then analyzed by filtering the data for minimum and maximum values.