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GENERAL CONSIDERATIONS FOR GENE EXPRESSION ANALYSIS IN YEAST USING DNA MICROARRAYS
General Considerations for Gene Expression Analysis in Yeast using DNA Microarrays
Overview
This document describes general considerations for gene expression analysis of gene expression in Saccharomyces cerevisiae using DNA microarrays. DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide or other solid substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization of the prepared cDNA to the spotted DNA on the microarray. Because the yeast genome consists of approximately 6000 genes, the expression profile of the entire genome can be analyzed with a few microarrays. This document offers useful advice on the setup, execution, and analysis of microarray experiments in yeast.
Procedure
I. Experimental Setup
A. Consideration of the Experimental Parameters
1. Choose yeast strains to compare that are as identical as possible (same genetic makeup, mating type, and minimal auxotrophies). Strains of S. cerevisiae vary widely in genetic makeup. This is often referred to as a "strain background." For example, different strains will carry different mutations in "marker" genes that allow for the selection of yeast strains based on an inability to grow on media lacking key supplements (e.g., adenine, histidine, uracil, etc.; see Protocol ID #9073). Additionally, the common laboratory strains in use today, such as W303 and S288C, also carry many multiple differences in the DNA sequences of their genomes. Because of these sequence differences, the use of strains that have different genetic backgrounds will create differences in hybridization to the microarrays that do not correspond to changes in gene expression due to the experimental variables under study.
2. Perform pilot studies to determine the optimal growth conditions, "dosage" of the stimulus, and appropriate time points for collection of cells. The pilot experiments should be designed to minimize any factors that are unrelated to the stimulus applied to the cells (e.g, diauxic shift, heat shock, hypoxia, etc.). Determine the optimal media, culturing conditions, and temperature for growth of cells. Also, expose the cells to a series of concentrations or variety of conditions of the stimulus.
B. Consideration of the Experimental Variables
1. It is preferable to limit the experimental variables in any one analysis. Compounding or overlooking experimental variables can confound analysis of the results. A common oversight is to use cells that are progressing through "diauxic shift." Diauxic shift occurs when cells deplete the media of glucose and alter their metabolism to account for the low carbon source conditions. The expressions of thousands of genes are affected by this simple event.
2. Note that certain stimuli will have pleiotropic cellular effects. For example, growth of cells in high sodium concentrations will alter the osmotic conditions within the cell, affecting the functions of cellular components as diverse as mitochondria to DNA replication.
3. Standardize a rapid, reproducible method to collect cells at the appropriate time points. Avoid extensive cell handling procedures. If there are multiple steps in the collection and processing (i.e., multiple washing steps and pauses before storage or RNA isolation), cells may suffer starvation conditions (e.g., hypoxia). Variations in the collection process should also be avoided.
4. For stimuli that consists of a substance (e.g., drug) suspended in a carrier solution, remember to include a carrier control.
C. Consideration of the Proper Reference
1. Choose a reference sample that will achieve uniform hybridization to the microarrays. Because the manufacturing process of the microarrays will inevitably yield variations in the amount of DNA contained within each "spot," a reference sample should be included to enable the normalization of the hybridization signals. The expression level for each gene can be normalized by computing the ratio of the experimental sample hybridization to the reference sample hybridization. This is achieved by labeling the experimental sample cDNA with a fluorescent molecule that fluoresces one color (e.g., green) and the reference sample cDNA with a fluorescent molecule that fluoresces a different color (e.g., red). Examples of reference samples include genomic DNA (no need to create cDNA), total RNA of cells from an unrelated experiment, RNA from cells just before exposure to the stimulus (i.e., time point "zero"), a pool of a portion of all of the RNA samples recovered from the experiment, or RNA from the control sample.
2. Use the same reference sample on each microarray in a given experiment. This will enable the comparison of the expression profile of multiple time points to each other.
3. A mathematical transformation example with genomic DNA as the reference sample: There will be one array for each time point including the zero time point. With the reference probes labeled with green and the sample probes labeled with red, the red-to-green fluorescence intensity ("R/G ratio") of each spot will be measured for each array. Divide the R/G ratios of each array by the R/G ratio of the zero time point array to derive the expression profile for each time point post stimulus. This is the equation for the calculations:
(R/Gt>0 array)/(R/Gt=0 array) = (RNAt>0/Genomic DNA)/(RNAt=0/Genomic DNA) = RNAt>0/RNAt=0
II. Execution of the Experiment
A. Preparation of Cells
1. Inoculate a culture of yeast cells so that the cells will proceed through two cell cycles. This will ensure that the cells are in logarithmic growth phase.
B. Condition of the Cells during the Experiment
1. Inoculate the starting culture with the subculture that has divided twice so that cells from all time points will be collected before the cells reach the diauxic shift. Note that reaching diauxic shift will depend upon the specific conditions of growth (e.g., rich versus minimal media, temperature, aeration, environmental stress, etc.).
2. If the experiment requires long intervals between time points, employ a chemostat, or other device, to maintain culture conditions. Work within the range of cell concentrations that permit logarithmic growth under the conditions chosen. For example, if the cell culture is reaching the point of diauxic shift, dilute back the culture with identical medium under conditions that are as comparable as possible (i.e., use pre-warmed media, work rapidly, etc.).
C. Observations during the Experiment
1. Record as much detail about the experiment as possible. It is often the case that the interpretation of the results depends on observations that are unrelated to the initial phenotype. Examples of observations to record are: the optical density at 600 nm wavelength of the culture, the cell number, the cell volume, the cell viability, the cell morphology, nutrient concentrations, and concentrations of the stimulus (if applicable). When recording this data, it is useful to photograph cell morphology. Additionally, freeze samples of the culture for later measurements. Finally, record any abnormalities or other unanticipated details during the experiment.
D. Sample Collection
1. Employ a consistent technique in the collection of samples. Collection by centrifugation will require approximately 3 to 5 min to complete, while collection by filtration onto a 0.45 μm filter will require less than a min. When employing either method, reproducibility is key.
2. Collect all samples under conditions that are as close to the growth conditions as possible. If cells are grown at room temperature, collection at room temperature avoids abnormalities introduced by a change in temperature. Collecting cells quickly will also minimize any temperature shock effects and hypoxic conditions.
3. Avoid common mistakes such as the following: Washing cells excessively induces stress response. Collection of cells on ice induces cold shock response. Lengthy collection times induce hypoxia. Variation in collection times results in variations in expression profiles.
III. Hybridization to Microarrays
A. Conditions of Hybridization
1. For optimum results, control all manipulations (e.g., temperatures, hybridizations, washes, times, etc.). Consistency is key for reproducibility.
2. Perform duplicates (or triplicates) of hybridizations for each time point. This will control for any variations in hybridization (e.g., variations in spotting, hybridization conditions, etc.).
IV. Data Analysis
A. Consideration of the Experimental Details
1. Keep in mind the experimental details during the analysis. Be aware of any pleiotropic conditions during the experiment, potential diauxic shift effects on the last time point samples, any strain background differences, the effects of cell cycle progression or arrest, and any secondary effects that the primary stimulus might exert on cells.
2. Attempt to identify responses that are likely versus responses that may be due to the undesired effects mentioned above. Comparison of the data to previous similar experiments will help determine specific responses. For example, metabolic changes during normal growth conditions may be identical between unrelated experiments and can be excluded based on the comparison.
3. Utilize any statistical program that provides ease of interpretation. Examples of software to analyze the data include hierarchical clustering, Self Organizing Maps, K-means clustering, Singular Value Decomposition, and other packages. Different methods have different strengths and weaknesses. Often the most thorough analysis involves multiple permutations of several of the computational methods mentioned above.
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