MATERIALS AND METHODS

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1.  TOTAL RNA ISOLATION from YEAST                              

       (complete protocol)

Growing Yeast Cultures

• Four yeast cell cultures were grown the day of the first experiment (see RNA ISOLATION and ANALYSIS).

• 50 mL of each yeast culture was plated on an agar plate (labeled accordingly) and grown overnight. The plates were labeled: Δzms1, Δzms2, Δzms1/Δzms2, or WT.

• Yeast was obtained from each plate and placed into a flask (labeled accordingly) containing 50 mL of SD growth media; the media appeared cloudy. The cultures were incubated at 30° C overnight.

• The following morning:

    1)        Cuvettes were set-up with 2.5 mL of media (the tops of the cuvette were labeled according to the

             strain).

    2)       A blank was set up using 3 mL of media

    3)       The flasks were removed from the shaker and gently shaken to resuspend the yeast. Then, ~500 µL  

             of yeast were placed into the corresponding cuvette and then covered with parafilm.

    4)       The spectrophotometer was set to 600 nm and zeroed using the blank containing 3 mL of media.

    5)       The cuvette samples were inverted to mix and placed into the spectrophotometer to obtain the

             Optical Density; the initial absorbance value was obtained. OD values between 0.1 and 1 were

             desired.

Preparing Yeast Spheroplasts

• Yeast cells have cell walls that were enzymatically degraded to obtain cells without cell walls, or spheroplasts. Spheroplasts facilitate yeast cell lysis performed to isolate yeast cell RNA.

    3)      The 2 mL centrifuge tubes containing cells were spun at 8% for 3 minutes.

    5)      3 µL of mercaptoethanol was used in order to break the disulfide bonds during cell lysis.

• The 1 mg/mL of lyticase was used to degrade cell wall components; the 1 mg/mL was actually 4 mg of lyticase per 4 mL of sorbitol buffer.

    7)      The microcentrifuge was spun at 4% for 3 minutes.

    9)      One tube or 1.6 mL of sterile dH₂O was not used here. 

Isolating the RNA

• RNA is very sensitive and easily degraded by omnipresent RNases. It was therefore necessary to use sterile technique to prevent unwanted degradation of RNA. Gloves were changed as needed and table surfaces and instruments were wiped down with RNAZap, from Ambion, to remove RNases prior to conducting the following portion of the experiment.        

    14)    Keeping the cells on ice inhibits RNase activity.

    15)   The cells were centrifuged at 9.5% for 10 minutes.

    17)    Isopropanol has a high salt content and is used to precipitate nucleic acids.

    18)    Tubes were spun at 9.5% for 5 minutes.

    21)    Pellets were resuspended in 100 µL of Solution 3.

    22)    20 µL of Solution 4 was added to the samples; this solution contains a resin that has DNases 

           on it that will bind DNA but not RNA).

    24)    Tubes were spun at 9% for 2 minutes.

Quantifying RNA

• A nanodrop was used to determine both the concentration and purity of previously isolated RNA. Each sample was measured twice and the average value used for the purpose of this experiment.             

Checking the RNA for degradation using gel electrophoresis

• 2 µg of RNA were run on a 1.2% agarose gel at 100V for 30 minutes. A 1.2% gel was created using 0.3 g of agarose powder and 25 mL of buffer. Ethidium bromide was used in order to visualize the RNA bands using UV light.

2.  LABELING PROCEDURE for MICROARRAY ANALYSIS

           (complete protocol)

• To label the probes, the mRNA from previously isolated yeast cells must be reverse transcribed into cDNA using a poly-T primer. The poly-T primer has a 3 DNA capture sequence for each specific dye (red or green). 

• Once the mRNA has been reverse transcribed, the cDNA wil be mixed with Cy3 (green dye) and Cy5 (red dye); the probe will be red or green depending on which DNA sequenced is recognized. Ultimately, the probe will resemble the following:

5’  ----3DNACaptureSequenceTTTTTTTTTTTTTTTTcDNASequence----3’

• Extreme caution was used when handling RNA, as it is extremely vulnerable to RNases.

Reverse Transcription

• The goal of this procedure is to successfully label cDNA of Δzms1/Δzms2 mutant with red dye (Cy5) and cDNA of WT with green dye (Cy3).

    1)   RNA Reverse transcription was set up for each RNA sample.

• 5 µL of total RNA (containing mRNA, rRNA, and tRNA) was used

    6)   The dNTP mix was kept on ice to thaw.

    9)   In this step, a change in pH was used to degrade the RNA.

Concentrating the cDNA

• The goal of this procedure is to successfully label mutant Δzms1/Δzms2 with red dye and WT with green dye.

    9)      A total of volume of 8 µL was obtained; 2 µL of nuclease free water was added to bring the   

            volume up to 10 µL.

3.  2-STEP HYBRIDIZATION of the DNA CHIP USING the 3DNA ARRAY 350 PROTOCOL        (complete protocol)

Pre-Hybridization

• Salmon DNA (sperm) was used to indirectly block the microarray slide.

            4)   It is important to spin the slide as soon as it is removed from the SSC/SDS mixture to prevent

                   salt from drying on the slide; this will skew results.

Hybridization I

• A final volume of 58 µL resulted from the below mixture  .

10 µL of concentrated cDNA

2 µL of LNA dT Blocker (to prevent the poly-T primer from interacting with the array slide)

29 µL of 2X enhanced hybridization Buffer

17 µL of Nuclease Free Water

•  The entire 58 µL of mixture was transferred to the microarray surface. Slide number 13760727.

Washing I 

Hybridization II

• This step is light sensitive and must be performed in the dark.

Washing II

        17)     The tube was removed from the centrifuge and the back of the microarray slide was cleans  

                 using a kim wipe and ethanol.

        18)     Once clean, the slide was placed into a wooden box covered with aluminum foil to be later  

                 packaged by Dr.Rife.

4. DATA ANALYSIS

    Since James Madison University does not have the technology to scan microarray slides, slides were sent to Davidson College in Davidson, NC to be scanned. The resulting data was obtained from Davidson College. At JMU, ScanAnalyze was used to analyze the fluorescent data in grid format; the data was then transferred to Excel spreadsheets.

    The intensity of each fluorescent spot of interest was calculated by subtracting the background from the read intensity. The resulting mutant intensities were divided by corresponding wild type intensities to produce values that would be transferred into the biological/statistical program MagicTool. MagicTool was used to create a log₂X expression scale of the data as well as to standardize the dye intensities. Statistical analysis was used to determine which genes were up and down regulated.

    The specific gene names and corresponding biological and molecular functions were obtained from the GCAT website.

The following table represents the strain and corresponding color of mutant or wild type genes.

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