This protocol will allow you to isolate enough total RNA from yeast to label for our microarray experiments.  There are five basic steps we will go through--1.  Growing the Yeast Cultures, 2. Preparing Yeast Spheroplasts, 3. Preparing Yeast RNA, 4. Determining the concentration of yeast RNA, 5. Checking the RNA for degradation using gel electrophoresis.

Remember as you step through this procedure that there are RNases (Enzymes that break down RNA) everywhere.  Your hands and breath are two major sources. You should wear gloves throughout these procedures and avoid using your gloves to touch your skin or hair during the procedures.  Do not directly breathe or talk over the tubes. Do not have anything unneeded on your benches and think the protocol through before you begin. 

Your benches should be washed down with a special detergent called RNAZap (Ambion) that will help get rid of Rnases before you start the protocol.

Cultures of wild-type and yeast containing various mutations will be started the day before the experiment. On the day of the experiment the cultures will be assessed to make sure both the mutant and wild type cells will have the same amount of yeast in them by the time class starts.    Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class. 

Yeast are grown very similiarly to bacteria.  Like bacteria, they have different phases of growth. The three phases of growth are called the lag phase, log phase and stationary phase. Yeast start off in something called lag phase, its called the lag phase because it takes awhile for cells to turn on the genes they need to start growing. Then they begin to double exponentially, this is called log phase. Finally, when the nutrients in the media are used up, the yeast enter into what is known as stationary phase where their growth stops.     Reading the absorbance of a culture on a spectrophotometer at 600 nm is the way in which you can determine what phase of the yeast growth cycle you are in and how many cells you have. The results of these readings are measured as optical density units or O.D.   We want to harvest our yeast cells in late log phase because we want yeast that are still growing and the maximum amount of yeast we can have in our culture.  Haploid yeast cells only contain approximatley 1.2 pg of RNA per cell so we need a lot of cells to get the ug of RNA we need to work with!  Below are some results from a growth curve experiment done by Bobby East, a student in a previous class. He started both a wildtype and a ZMS2 knockout culture at an O.D. of 0.3. How many hours does it take before the yeast in his experiment stop growing and enter stationary phase? If we start our yeast off at an OD of 0.3 at 8:30 a.m. in the morning, will they be in late log phase at 2 p.m. when we harvest them for our class? Let me know if you think we should modify our timing?

Growth Time for Yeast (Minutes)	OD 600 of WT	OD 600 of ZMS2 Knockout
0	0.3	0.27
231 min. (~4 hours)	0.579	0.537
281 min. (~4.5 hours)	0.813	0.744
326 min. (~5.4 hours)	0.909	0.912
356 min. (~6 hours)	0.957	0.942
386 min. (~6.5 hours)	1.02	1.03
416 min. (~7 hours)	1.06	1.07
466 min. (~7.7 hours)	1.09	1.09

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded.  These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don't want to lyse the cells before they are in this controlled environment.

1) Place 50 ml tubes containing 30 ml of the yeast cultures at the appropriate O.D. reading in the centrifuge next door (Make sure it is balanced). Centrifuge at 2,500 RPM (1,500 x G) for 5min to pellet the cells.  Please share the centrifuge as there is only one.

The next 3 steps are called a wash--they serve to move the cells from the growth media into a solution that is correctly buffered for the next procedure.
2) Remove tubes from centrifuge. Carefully pour supernatant into the liquid waste container. Try not to disturb pellet.  Add 1ml of Potassium Phosphate/Sorbitol buffer  (1.2 M sorbitol, 10 mM potassium phosphate, pH = 7.2) and resuspend the pellet by pipetting.

3)  Transfer the cells from each conical tubes into separate 2 ml microcentrifuge tubes (these are colored). Spin at 80% for 3min.

4) Pour off supernatant (without disturbing pellet). Remove remaining supernatant with pipet Resuspend pellet in 1ml Potassium Phosphate/Sorbitol buffer.

5) Take tubes to the fume hood. Add:

• 3µl b mercaptoethanol (a reducing agent that smells very bad)
• 320 µl of 1 mg/ml lyticase  (enzyme that degrades cell wall components)

6) Place tubes in shaker at 30°C for 15min.
The cells are now spheroplasts. Without a cell wall they are alive but strucutrally much weaker.

Right before breaking the cells  we must get rid of the lyticase and b-MeOH.
7) Spin in microcentrifuge at 40% for 3 min. Remove supernatant by pipetting (discard in hood to contain the smell).

8) Add 500µl potassium phosphate/sorbitol buffer. Repeat step 7 (one time).

9) While centrifuging get an ice bucket  ready containing:

2 microfuge tubes containing glass beads,1 tube of Solution 1 (1.6ml),1 tube of Solution 2 (400µl),1 tube isopropanol (1.6ml),1 tube of Solution 3 (400µl)

The RNA isolation will be done using the RNAsafe kit from Qbiogene. Solution 1 and 2 contain ions and detergents that will inactivate cellular RNases that are released when you lyse the cells and help to break through the cell membrane. Eventually you will spin the mixture down and get rid of all of the proteins in the solution and the 900 ul you take out in step 15 should contain mainly nucleic acids. The next few steps help to remove the DNA from the solution.

10) Resuspend pellets from step 9 in 800ul Solution 1 by gently flicking tube.

11) Pour glass beads from tube in icebucket into tube containing cells.

12) Add 200µl of Solution 2 to each cell+bead tube. Solution 1 and 2 contain buffers and chaotropes that will help stabilize the RNA in solution).

13)  Vortex the cells on high for 10 cycles of vortexing / ice alternatations ( 1 cycle = 30 seconds of vortexing / and then 30 seconds on ice).  This step breaks open the yeast by brute force.

14) Incubate tubes for 2min on ice.

15) Centrifuge at 95% for 10min.

16) Transfer 900µl of supernatant to a clean tube (if you can't get this much it is okay just adjust the isopropanol amount in the next step).

AVOID ALL DEBRIS at the bottom of the tube as well as 'gunk' layer at the top. It is better to take less than 900ul and avoid 'gunk'. The debris contains membranes, unbroken cells, and proteins. Since the yeast contain cellular RNases (a protein) it is important to separate them from your RNA.

17) Add 900µl isopropanol to each tube. Mix by inverting the tubes. Then allow to set at RT for 2 min.

18) Centrifuge tubes at 95% for 5min to pellet the RNA precipitate.

19) Carefully decant the supernatants by inverting a tube and giving it a decisive flick to remove all the supernatant.

20) Spin the tubes again for 30 sec and remove any residual supernatant with a pipetman.

21) Resuspend each pellet in 200µl Solution 3. Make sure pellet is resuspended--flick it, vortex it, make sure the pellet is gone.

22) Add 40µl Solution 4 to the samples. Mix by vortexing for 10sec.(Solution 4 contains a resin that binds DNA but not RNA)

23) Incubate in your rack at room temp for 5min.

24) Spin 90% for 2min.

25) Carefully MOVE the SUPERNATANT to the correct clean tube.  Make sure it is well labeled as you will need to store this in the freezer until next week.  Always keep your RNA on ice!!!

The concentration of RNA is read by measuring the absorbance of a sample at A260 on a spectrophotometer.  The RNA sample is placed in a quartz cuvette in order to read the optical density (OD). Common glass and plastic will also absorb at this wavelength so special quartz cuvettes that do not absorb light at this wavelength are used to measure the absorbance of the RNA. Be very careful with these, as they are very expensive.

You will need to use it to determine the concentration of your RNA.  Scientists have determined an absorbence coefficient to use to determine the concentration of RNA in your sample preparation.  An RNA with the concentration of 40 ug/ml has an optical density of 1 at A260.  When you get the reading of your sample you can use this information to find out how much RNA you have.

To get your reading put 20 ul of your sample in 1 ml of water and place it in a quartz cuvette.    Once you obtain your reading the equation should be used to help you determine your sample concentration
 

The dilution factor in this case would be 1000/20 = 50 since you dissolved 20 ul in 1000 ul of water.

Scientists often measure the absorbence of their RNA sample at A280 as well as A260. Both proteins and nucleic acids absorb light at 280 nm. By obtaining the ratio of A260/A280 scientists can thus get an idea of the purity of their RNA sample. For pure RNA without a lot of protein this ratio should be 1.9-2.2.
 

You will use 2 ug of RNA to run on a 1.2% agarose gel at 100 V for 1/2 hour.  

To each sample of RNA add the appropriate amount of loading dye and 1 ul of Ethidium Bromide

Several groups can run on one gel.  We are looking for the ribosomal RNA bands.  They should look very distinct and not degraded for the best quality RNA.  See the picture below for an example using rat RNA.

1 = Degraded RNA

2= Good RNA

3 = Good RNA