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Isolation of Total RNA from Yeast
(This protocol adapted from Dr. Karen Bernd, at Davidson College, N.C.)
Objectives:
1. To learn how to work with yeast and extract Total RNA from them.
2. Understand the importance of RNases and how to protect RNA samples.
3. Quantitate and check purity of nucleic acid samples
4. Understand the difference between fermentation and respiration in yeast
5. Understand the steps required in experimental design and planning
READ THIS PROTOCOL CAREFULLY. MAKE SURE YOU ARE
PREPARED TO ASK ANY TECHNICAL OR THEORETICAL QUESTIONS
BEFORE WE BEGIN THIS EXPERIMENT. IF IN DOUBT ABOUT
ANYTHING, ASK!
Isolation of total RNA from yeast
RNA expression is one way of measuring gene activity. As genes are turned on,
mRNA levels for those genes increase, generally resulting in increased protein
expression. While cells have many ways of regulating proteins, differences in the
functional state of the cell correlates directly with changes in mRNA levels. The
cells use these ‘new’ proteins to respond to the current environmental conditions
in the cell.
In this experiment, we will monitor the yeast’s response to decreasing
concentrations of glucose in the media as the yeast are forced to live under less
favorable conditions. In rich media (i.e. high in glucose) the yeast metabolize the
sugars to ethanol through anaerobic fermentation, but under less favorable
conditions (little or no glucose) , they start metabolizing ethanol by aerobic
respiration. This process of changing ‘food sources’ in yeast is called the diauxic
shift. During the diauxic shift the yeast undergo substantial changes in gene
expression related to the many biochemical pathways involved in the different
methods of metabolism. We will try to gain insights into gene expression by
examining total mRNA levels in yeast at critical time points during the shift using
microarray technology. The first step in this process is to obtain yeast cultures at
time points that represent the changes that occur during the diauxic shift, starting
with yeast that have been grown in the presence of high glucose (i.e. the positive
control) and subsequently in lower glucose concentrations. Your job is to isolate
the total RNA at one time point. We will share our data, to get a complete time
course of the diauxic shift when we complete the microarray analysis for each
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time point later in the semester.
In order to characterize the diauxic shift in yeast, you will be isolating total RNA
from cultures of YEAST (STRAIN DBY10009) –a generous gift from the Botstein
Lab, Princeton University, grown for different lengths of time . This particular
strain of yeast has been completely sequenced. Later on, we will analyze gene
expression of the ~6000 genes in yeast and try to correlate differences with their
growing conditions. Your results for the future microarray work will depend on
how well you complete this initial phase of the experiment. Make sure you work
CAREFULLY.
RNA is extremely sensitive to degradation by RNases. How carefully you
handle your samples and transfer solutions will have a huge impact on your
yield.
You must get rid of RNases. Your objective is to isolate a large quantity (perhaps
100 whole micrograms) of high molecular weight, undegraded total RNA. Read
all instructions before touching anything. Make sure you have cleaned your bench
space before beginning.
The most important step in RNA isolation is to remove as many sources of RNases
from your work area as possible. RNases are enzymes whose entire purpose is to
degrade RNA. They are very stable, low molecular weight proteins that can
withstand high temperature and they are EVERYWHERE (yes, be paranoid!)
ANY contamination with RNase will destroy your sample and pretty much wreck
your day, experimentally speaking.
Extra caution now will save you much work later. The main source of RNase
contamination is your hands. Since it is out of the question to bake your hands at
210° for 15 hours to inactivate the enzyme, you must do everything you can to
eliminate contact between your skin, things your skin has touched, and your
precious samples. Clear your bench of all but the bare essentials. Once you have
broken open the cells the RNAs are released and are susceptible to degradation by
RNAases. Wear gloves and wash down your entire area with 'RNase ZAP'
(special detergent that helps control RNases a bit.)
All microfuge tubes and pipet tips have only been touched with gloves and have
been sterilized extensively.
The protocol is not difficult but you must be organized and careful.
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Materials:
Yeast (Saccharomyces cerevisiae – Strain DBY10009)
YPD Media (autoclaved)
10 g/L Yeast Extract
20 g/L Peptone
20 g/L Dextrose
Lyticase (20Units/mg) (incubate @ 35ºC)
Isopropanol
50 ml Conical tubes
1.6 ml eppendorf tubes
200 ul & 1000 ul RNase Free pipet tips
Pipetmen
1.2 M Sorbitol, 10 mM KPhos pH 7.2
Potassium Phosphate (mw = 136.09 g/mol)
Sorbitol buffer (mw = 182.17 g/mol)
Total RNA Safe Kit (www.mpbio.com)
RNAse ZAP
RNA Safe Kit
Glass beads (0.5 mm in diameter – autoclaved)
Vortex Mixer
30ºC Heating Block filled with deionized water
Swinging bucket centrifuge @ 4ºC (G-309)
UV Spectrophotomer (in Chemistry Lab -- Read Absorbances at 260 and 280 nm)
Cuvettes (1 ml sample volume)
1.2% Agarose Gels
10X Nucleic Acid Sample loading dye
Wide Range Markers
Gel Running Bufffer (1 liter electrophoresis buffer 1x TBE)
Ethidium Bromide
Power Supply
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Preparing yeast cells by making spheroplasts
Yeast cells are grown in YPD media at 30º C in a shaking incubator. Yeast
cultures grow exponentially until all media is exhausted. Growth of the yeast
cultures in monitored by optical density, (Absorbance at 600 nm).
Yeast are grown very similarly to bacteria. Like bacteria, they have different
stages of growth. Scientists divide these stages of growth into early-log phase,
mid log-phase, late log-phase, and the stationary phase. These phases are based on
the amount of yeast that are around in the broth and how quickly they are able to
grow and divide based on nutrient use. Reading the absorbance of the sample at
600 nm is a way in which you can determine what phase of the yeast growth cycle
you are in and how many cells you have. Here is a chart that will show you how
this relates to yeast growth. You should record the O.D. of the class cultures. early log-phase
mid log-phase
late log-phase
stationary phase
OD 600
cells/ml
< 0.4
0.4 - 1.7
1.7 -6.6
> 6.6
< 10E7
1-5 x 10E7
5 x10E7 - 2 x 10E8
2 x 10E8
Haploid yeast cells contain approximately 1.2 pg of RNA per cell.
Predict approximately how much RNA you should isolate.
The volume of culture for each time point was adjusted so that each sample (time
point) contains the same amount of yeast. The cultures were inoculated and grown
for 9 hours, then samples were withdrawn from the culture every 4 hours. The
yeast cells were pelleted and then flash frozen and stored at -80ºC. Your group
will receive yeast cells that were grown for a certain period of time (9 hrs, 13 hrs,
17 hrs or 21 hrs). We expect gene expression in the yeast to change over time
because glucose is being depleted.
Yeast contain a hard cell wall. To improve our RNA yields we need to breakdown
the cell wall so that the cells may be lysed more easily. Spheroplasts are yeast cells
where the cell wall has been enzymatically degraded.
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1) Each group receives a yeast culture that has been collected at one of the above
time points (9 hrs, 13 hrs, 17 hrs or 21 hrs).
2) Label your yeast sample according to the time point you’re given so that you
can identify your tube. Record the OD600 of your sample.
3) Get your supplies ready: yellow and blue tips for pipetmen,
1.6 ml Microfuge tubes, and Potassium Phosphate/Sorbitol buffer.
The next 3 steps are called a wash--they serve to move the frozen cells into a solution
that is correctly buffered for the next procedure.
4) Add 1.0 ml of Potassium Phosphate/Sorbitol buffer to your yeast pellet. Let the
yeast thaw on ice for at least 5 minutes. Resuspend the pellet by gently pipetting.
5) Label one 1.6 ml microfuge tube. Transfer the cells from the 50 ml conical
tubes to the microfuge tube.
6) Place the microfuge tubes in the microcentrifuge (balancing tubes). Spin at
11,200g for 3min @ 4ºC.
7) Pour off supernatant (without disturbing pellet). Remove any residual
supernatant with a pipetman. Resuspend pellet in 1.0 ml Potassium
Phosphate/Sorbitol buffer.
8) Take tubes to hood. Add:
3µl -mercaptoethanol (a reducing agent that smells very bad)
320 µl of lyticase (enzyme that degrades cell wall components)
9) Place tubes in a heating block water at 30°C (check TEMP!!) for 15min.
The cells are now spheroplasts. Without a cell wall they are alive but structurally
much weaker. Care must be taken so that the cells don't burst before you want them
to. (How could the buffer they are in help keep them intact?)
NOTE: yeast will repair their cell wall if the enzyme is removed so we cannot just
keep a stock of 'wall free' yeast.
You must now get the cells ready for the next set of steps by washing away the
lyticase and -mercaptoethanol.
10) Spin in microcentrifuge at 5600 g. Remove supernatant by pipeting (discard in
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hood to contain the smell).
11) Add 500µl potassium phosphate/sorbitol buffer. Repeat step 10 (one time).
12) While centrifuging make sure your ice bucket contains:
1 microfuge tube containing glass beads
Solutions 1-3 are part of the Total RNA Safe Kit
(Solutions 1 and 2 contain RNA stabilizing chaotropes and buffers. The solutions
solubilize proteins and RNA and enable DNA and cellular debris to be precipitated.
Solution 4 contains a resin that binds DNA but not RNA
1 tube of Solution 1 (1.6ml),
1 tube of Solution 2, (400µl)
1 tube isopropanol (1.6ml),
1 tube of Solution 3 (400µl),
1 tube of sterile dH2O (1.6ml)
Clean RNA isolation area with 'RNAse ZAP' solution
(1 spray bottle for lab)
Isolating RNA
FROM THIS POINT ON DO NOT TOUCH ANYTHING ON YOUR BENCH
WITHOUT WEARING GLOVES.
DO NOT 'DOUBLETOUCH' TIPS
DO NOT WALK AROUND WITH OPEN TUBES
DO NOT LAUGH, TALK, OR BREATHE OVER OPEN TUBES
(To quote the movies "be afraid--be very afraid")
13) Resuspend pellets from step 12 in 800ul Solution 1 by gently flicking tube.
14) Pour glass beads from tube in ice bucket into tube containing cells.
15) 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).
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16) Using the vortexer, vortex the cells on high for 10 cycles of 30 seconds
vortex/30 seconds on ice. This step breaks open the yeast by brute force. (Why do
you think the glass beads are included? Why include cycles on ice?)
17) Incubate tubes for 2 min on ice.
18) Centrifuge at 13,500 g for 10 min.
19) While tubes are spinning- label a clean 1.6 ml eppendorf tube.
20) Transfer 900µl of supernatant to the clean 1.6 ml eppendorf tube.
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.
21) Add 800µl isopropanol to your tube. Mix by inverting the tubes for 2 min.
The alcohol reacts with the RNA (which is a salt) and causes it to precipitate out
of solution.
22) Centrifuge tubes at 13,500 g for 5 min to pellet the RNA precipitate.
23) Carefully decant the supernatants by inverting a tube and giving it a decisive
flick.
24) Spin the tubes again for 30 sec and remove any residual supernatant with a
yellow tip and P200 pipetman.
25) Resuspend your pellet in 200µl Solution 3. Make sure pellet is resuspended-flick it, vortex it, make sure the pellet is gone.
26) Get Solution 4 from me. Add 40µl to the sample. Mix by vortexing for 10sec.
27) Incubate in your rack at room temp for 5min (return solution 4 to me)
Solution 4 contains a resin that binds DNA but not RNA
28) Spin 90% for 2min.
29) Label a new tube for your purified sample --include identifying marks, date,
time point etc. since this tube will be stored in the freezer with other samples.
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30) Carefully MOVE the SUPERNATANT to the correct clean tube.
THIS IS YOUR RNA.
Place the tube on ice!
Your RNA should always be stored on ice or frozen to slow degradation (why
would cold slow degradation?)
Question: What types of RNA have you just isolated? (how many different kinds
of RNA are in a cell?)
Quantifying RNA
When you perform the microarray analysis later it will be important that the total
amount of RNA at each time point is known. Quantification of RNA using
spectrophotometry allows you to determine how much RNA you have isolated as
well as how free of protein contaminants it is. (hint: A well-prepared lab group
might have 2 members complete this part while the other 2 prepare for the next
section)
1) Make sure that UV spectrophotometer is on. The UV lamp must be turned on
and must have 10 min to warm up. The life of this lamp is GREATLY reduced if
you turn it on and off. During this lab just leave it on. When quantifying RNA try
to coordinate with other groups.
2) Get 2 quartz cuvettes from me. Hold cuvettes by the edges--fingerprints on the
flat surface will cause inaccurate readings. One cuvette is for your sample. A
'blank' cuvette will be left by the spectrophotometer.
3) Place 996µl of sterile distilled water into each cuvette. Add 4µl of your yeast
RNA into one cuvette.
What dilution of RNA did you just perform? A one in __________dilution. This
means that you diluted the sample by a factor of ________ (same # as above).
4) Hold a piece of parafilm across the top of the cuvette and mix the contents by
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inversion.
5) Take cuvettes to spectrophotometer. The UV spec will scan your sample in the
range from 190 nm to 700 nm. Place a BLANK cuvette (containing water only) in
to zero the spectrophotometer. Place your sample in the spectrophotometer to get
your readings. Use the cursor to obtain the absorbance at 260 nm and 280 nm.
SAMPLE Name ___________
OD260=__________
OD280=__________
7) Calculate the 280/260 ratio. A 'very clean' sample will have a 280/260 ratio of
between approximately 0.4 to 0.55
SAMPLE 280/260=
8) Calculate the concentration of RNA in each sample using the following
equation. (Note that the equation only uses the OD260.)
[µg/µl] = [(OD 260) * (Dilution factor)]/24
bold= conversion coefficient for RNA
italics= you calculated the dilution factor in #3
SAMPLE CONCENTRATION:
AFTER you have calculated the sample concentration add 4µl of RNAsin to each
sample. RNAsin inhibits RNAses (and keeps your 'RNAs…in…'). This compound
should help keep your samples intact.
Checking RNA Integrity
The quality of your RNA can be checked by agarose gel electrophoresis
Prepare a 5 ug sample of your RNA for analysis by 1.2% Agarose gel.
Coordinate with other groups to load samples (one sample from each time point)
and markers on the gel.
Run the gel at 120 mV for approximately 20 minutes until the two markers divide
the gel into roughly equal thirds
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Stain the gel with ethidium bromide.
Note the position of bands on the gel and the intensity of the staining. (Does your
sample look like the ones shown the sample gel below?)
1 = Degraded RNA
2 = Good RNA
3 = Good RNA
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Total RNA SAFE KIT Product Information (for your reference)
Samples should be prepared with concern to prevent degradation of RNA by RNases.
Immediate flash freezing of samples in liquid nitrogen after collecting or harvesting is the
most effective means to virtually stop RNA degradation. Freezing also aids in the release
of RNA from cells because cellular membranes are ruptured by ice crystals.
Solutions 1 and 2 contain RNA stabilizing chaotropes and buffers. The solutions
solubilize proteins and RNA and enable DNA and cellular debris to be precipitated.
The RNA is precipitated from the clarified supernatant with Isopropanol. Traces of DNA
are catalytically removed and the RNA is further purified with a proprietary matrix
suspension. The resulting RNA is DNA free and application ready.
BIO 101’s novel RNA isolation procedure precludes the use of Guanidine and harmful
organic solvents such as Phenol and Chloroform. The only organic solvent required is
user-supplied Isopropanol.
The resulting RNA is an efficient substrate for RT-PCR*, RPA, RNA blotting, primer
extension, poly A+ RNA selection and Differential Display.
* PCR process is covered by Hoffman LaRoche