Abstract Introduction Methods Results Discussion References
Total RNA Isolation from S. cerevisiae Yeast
Total RNA isolation was performed to isolate enough total RNA from yeast to label the microarray experiments. ZMS2 knockout mutant and wild type cultures of S. cerevisiae were grown overnight. Cultures were harvested during the mid log-phase when the O.D reading at absorbance of 600nm was between 0.4-1.7. Once the cultures were obtained, the first step was to prepare yeast spheroplasts by weakening the cell wall. Throughout the entire protocol, RNAzap was used to create sterile conditions free of RNases. The isolated RNA was quantifies using spectrophotometer reading at absorbance of 260nm and 280nm. The isolated samples were run on Agarose gel to ensure that the isolated RNA is not degraded before progressing to the next step. 2μg of RNA was loaded on 1.2% Agarose gel instead of 10μg as indicated in the protocol (See detailed protocol: Total RNA Isolation From Yeast and RNA Quality Control).
Probe Construction
The total RNA isolated from the each of the mutant and wild-type samples were reverse transcribed into cDNA, which is a more stable form. The wild-type control was labeled with Cy 5 (red) dye whereas the ZMS2 knockout mutant was labeled with Cy 3 (green) dye. After labeling both samples with dyes, the samples were mixed and stored in the freezer until the hybridization step. (See detailed protocol: Labeling Procedure For Microarray Analysis).
2-Step Hybridization of the Microarray Slides
The cDNA was then hybridized onto the microarray slide (Slide# 13697519) containing entire yeast genome. The first step in hybridization involved removing excess salts and denaturing oligos that might be present on the slides. The slides were then pre-hybridized with sonicated salmon DNA in order to prevent non-specific binding of cDNA probe to the yeast genome. First hybridization step involved treating the slides with 10µl of concentrated cDNA, 2 µl of LNA dt Blocker, 25 µl of 2X Formamide Hybridization Buffer, and 13 µl of Nuclease Free Water. During step 11 of 2nd hybridization, the tubes were incubated at 45°C instead of 55-60°C. The following hybridization step involved wash steps. In order to reduce the dye degradation, all the hybridization steps were performed in dark room (See detailed protocol: 2-Step Hybridization of the DNA Chip Using the 3DNA Array 350 Protocol).
Figure 1. Steps to Microarray slide preparation
Microarray Data Analysis
Gridding:
Data from microarray analysis was observed and thrown out due to uninterruptible results. Slide 104 from the 2004 microarray project was used for analysis. The top portion of the array consisting of 16 grids each containing 21 rows and 22 columns was gridded using ScanAlyze software. Detailed methods and procedures can be observed at Scanalyze Guide . Column spacing was set at 200um, row spacing at 200 um, spot width at 13 and spot height at 13.
Segmentation:
Data was segmented by creating a ratio of mutant signal over wild-type signal using EXCEL. A God-list was attached to label all genes on the array. Data was then processed, all negative ratios and zero ratios were thrown out. Segmented data was then combined with the gridding of the bottom portion slide 104 and duplicates of both top and bottom grids processed by other lab groups. All the ratios were then converted into their log base 2 value to reduce outliers.
For further information on the software and programs used please visit: http://www.bio.davidson.edu/
RNA Viability Confirmation:
The original ZMS2 knockout mutant and wild type isolated RNA was thawed and run on an agarose gel. 2µg of each RNA sample was combined with 2µL of 6X loading dye and 1µL of ethidium bromide (5mg/mL). The samples were separated on a 1% agarose gel. (See detailed protocol: Total RNA Isolation From Yeast and RNA Quality Control).
MAGIC Tool
All data was then imported into MAGIC Tool for further analysis. Data was log2 transformed and genes were searched using the parameters of 1 standard deviation above or below the mean expression level (approximately -1.45) for induction or repression. This produced too many genes to analyze, therefore 2 standard deviations was set as the overall cut-off criteria. Genes were searched for minimum expression values to be greater than 0.9408, producing 5 genes induced in all 4 trials. Genes were searched for maximum expression values to be less than -4.2196, producing 10 genes repressed in all 4 trials. These genes were then further analyzed for function and relevance to ∆ZMS1.
For further information on how to use MAGIC Tool please visit: Magic Tool Information
Expression Determination:
Expression ratios of genes for the methionine synthesis were determined using scanalyze. Slide 104, 106 and 7522 were analyzed. Ratios were log transformed by base 2 using Microsoft excel. The averages and standard deviations were found for each slide. Ratios were subtracted from the average and then divided by the standard deviation to determine the number of standard deviations the expression ratio is from the average. Genes with the expression ratio greater than +1 standard deviation were considered to be induced and ratio less than -1 standard deviation away was considered to be repressed.