| Methods & Materials | ||
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Introduction Microarray Technology Oxidative Stress in Yeast Hypothesis & Predictions Experimental Precedent
»Materials & Methods
Results
Discussion
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Growth and Preparation of Yeast Cultures and Isolation of RNA from Yeast Spheroplasts Yeast cultures of wild-type and knock-out mutant strains; DZMS1, DZMS2, and DZMS1 and DZMS2, were grown in nutrient broth. Prior to the experiment, the growth phase of cultured yeast was determined by reading absorbance at 600 nm. Only cultures in mid- to late log-phase, with an optical density (OD) of 1.5 to 2.5, were utilized, to ensure that growing healthy cells were being used. Yeast were transformed into spheroplasts, cells that have had their cell walls enzymatically degraded, to ease both cell lysis and extraction of total RNA, using an RNAase-free procedure and beta-mercaptoethanol and lyticase. The spheroplasts were then lysed and total RNA was isolated from the cell using Qbiogene (Solon, Ohio) RNAsafe kit reagents and RNAase-free technique. RNAZap (Ambion Inc., Austin, Texas) was used to remove any RNAases in the laboratory environment. Following isolation, the supernatant was stored frozen in an RNAase-free microcentrifuge tube. James Madison University; BIO 480 Lab Protocol
Quantification of Isolated RNA from Yeast Spheroplasts Isolated RNA was quantified using a Nano-Drop spectrophotometer and 2 mL of the isolated RNA sample. The absorbance at 260 nm (A260) and the absorbance at 280 nm (A280) were recorded to determine RNA purity. The concentration of RNA extracted was also recorded, however, RNA concentration can also be determined with the following equation: A260 x [(40 mg/1000 mL)/1 OD unit] x dilution factor = concentration in mg/mL RNA purity was determined by calculating the A260/A280 ratio. For pure RNA, in which there is not a lot of protein, the ratio is usually within the range of 2.0 – 2.2. James Madison University; BIO 480 Lab Protocol
Examining RNA for Degradation using Gel Electrophoresis To determine whether there is RNA degradation within the sample, 10 mg of RNA was run on a 1.2% Agarose gel. Using the concentration of RNA determined (see Quantification of Isolated RNA from Yeast Spheroplasts), the appropriate volume of isolated RNA was added to the lane. Gel was run in electrophoresis box at 100 V for one half-hour. The gel was analyzed using BioLab Chemiluminescent camera following electrophoresis. James Madison University; BIO 480 Lab Protocol
Preparation of Microarray-Labeled Probes Isolated RNA from wild-type and mutant strains was labeled with green (Cy3) or red (Cy5) dye sequences, respectively. A dye reversal was done to ensure that the dyes did not influence the data, because red dye bound has higher intensities compared to an approximately equal amount of green dye bound. The microarray labeled-probes were created using reagents from Genisphere (Hatfield, Pennsylvania) 3DNA Array 350 Detection Kit. The kit contained a RNA-Reverse Transcription (RT) primer that contains a dye-specific sequence off the 5’ end, ensuring that RNA isolated from wild-type and mutant yeast samples would yield differences in color when read off the microarray slide if differences in expression occurred. The RT primer was bound to the 3’ Poly-A tail of the isolated yeast RNA to produce dye-specific cDNA. The two reverse transcription products were combined in the same microcentrifuge tube, concentrated using YM30 Microconcentrators, and stored at -70°C until use. James Madison University; BIO 480 Lab Protocol
Hybridization of Labeled cDNA to DNA Chip using 3DNA Array 350 Protocol The Yeast DNA Microarray chip contains two copies of the 70mers from the yeast genome. Prior to hybridization, the DNA chip was pre-hybridized in sonicated salmon DNA to ensure specific binding of labeled-cDNA to the microarray slide. Hybridization of the DNA chip utilized reagents and protocol from Genisphere 3DNA Array 350 Detection Kit and the isolated and concentrated labeled-cDNA. The hybridized DNA chip was covered with a cover slip and incubated for at least twenty-four hours. After incubation, the dye capture reagents, containing complement sequences to the dye sequences of the hybridized cDNA, were hybridized to the DNA chip and incubated in a dark room overnight. Any unbound dye capture reagents were washed off after incubation in a darkened room and the slides were sent to Dr. Campbell (Davidson College, Davidson, North Carolina) for scanning. James Madison University; BIO 480 Lab Protocol
Collection and Analysis of Microarray Data using ScanAlyze and Magic Tool (v. 2.1) With the data obtained from the slides sent to Davidson College, we were unable to see many significant changes in gene expression, so microarray slides from 2004 were analyzed using computer software. Mutants were knock-out ZMS1 (DZMS1) and ZMS1/ZMS2 (DZMS1/DZMS2). The slides numbers used and their corresponding channels were; Slide #104, Channel 1 (green, wt) TR104_w595, Channel 2 (red, DZMS1) TR104_685; Slide #106, Channel 1 (green, wt) TR106_595, Channel 2 (red, DZMS1/DZMS2). Microarray data provided was in duplicates (n = 2). Following scanning, images were opened using ScanAlyze Software. Channel 1 was the signal from the green dye (wild-type). Channel 2 was the signal from the red dye (mutant). Gridding, overlaying spots for analysis on the image of the slide, and segmenting, assigning gene names for each spot, were done using the software. Any microarray slide errors, such as dust or streaks on the slide, were corrected by flagging any spots associated with the slide errors. Intensities and background values for all the spots were saved and opened on Microsoft Excel. Using Microsoft Excel Software, the intensity values from each channel were corrected by subtracting the background value from the intensity value. Any negative number was removed from the corrected value. Channel ratios were calculated by dividing the corrected channel 2 by the corrected channel 1. Any ratio values of zero were removed from the data prior to further analysis. A ratio that was greater than one indicated increased expression in the mutant, while a ratio less than 1 indicated decreased expression in the mutant. Ratios were saved as “.exp” files. Further analysis of microarray data was done using Magic Tool (v. 2.1) software. A new project was created and the microarray ratios “.exp” file was opened. The ratios were transformed using log base 2 to ensure that all ratios fell between -16 and 16. The transformed data was then standardized with a mean of zero and standard deviation of one to help correct for dye bias. The data was filtered to include only ratios that were less than -2.5 or greater than 2.5, which would indicate an approximate five-fold decrease or increase in expression, respectively. The filtering criteria ensured that housekeeping genes and non-biological noise were not considered statistically significant results. The data was then transferred to Microsoft Excel to determine the mean and standard deviation of each gene of interest. Gene that were considered to be significantly over-expressed or under-expressed, in addition to being greater than 2.5 or less than -2.5, had a standard deviation that was less than or equal to 10% of the mean: the standard deviation divided by the mean was less than or equal to 0.10.
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