Cell and Molecular Biology Laboratory
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Lab 5 Isolation of Total RNA from Yeast
(This protocol revised & adapted from Dr. Karen Bernd, at Davidson College, N.C.)
Objectives:
1.
2.
3.
4.
5.
To learn how to work with yeast and extract Total RNA from them.
Understand the importance of RNases and how to protect RNA samples.
Quantitate and check purity of nucleic acid samples
Understand the difference between fermentation and respiration in yeast
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 challenges within 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 throughout 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 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.
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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. Any contamination with RNase will destroy
your sample and pretty much wreck your day, experimentally speaking.
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.
Extra caution now will save you much work later. The main source of RNase
contamination is your hands. 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. So WEAR gloves!!!
The protocol is not difficult but you must be organized and careful.
A. Materials:
Yeast (Saccharomyces cerevisiae – Strain DBY10009)
YPD Media (autoclaved)
10 g/L Yeast Extract
20 g/L Peptone
20 g/L Dextrose
Lyticase (20 Units/mg) (store @ 4ºC, incubate @ 35ºC)
Sigma L4025 50 KU from Arthrobacter luteus
(lyticase is an enzyme that breaks down yeast cells walls)
BME (ß-mercaptoethanol)
RNAse ZAP
100% ethanol
Use ACS grade 100% ethanol or better. You will need 40 mL to add to the Wash
Solution 2/3 Concentrate, and 1.25 mL per sample for use in step D.3.
15 ml tubes with tight fitting lids (ie corning)
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)
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Ambion RiboPure – Yeast Kit (Cat #AM1926)
Ambion RiboPure™-Yeast is a rapid RNA isolation kit that combines disruption of yeast
cells with Zirconia Beads, phenol extraction of the lysate, and glass-fiber filter
purification of the RNA. It can be used to isolate total RNA from a variety of yeast
species. RiboPure-Yeast was extensively tested with Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Pichia pastoris,and Candida albicans.
Amount
Component
Storage
50
50
100
1.5 mL Screw Cap Tubes
Filter Cartridges
Collection Tubes (2 mL)
room temp
room temp
room temp
24 mL
2.4 mL
24 mL
95 mL
35 mL
50 mL
Lysis Buffer
10% SDS*
Phenol:Chloroform:IAA †
Binding Buffer ‡
Wash Solution 1‡*
Wash Solution 2/3 Concentrate
Add 40 mL 100% ethanol before use
4°C
4°C
4°C
4°C
4°C
4°C
5 mL
Elution Solution
4°C
80 g
550 µL
200 µL
550 µL
Zirconia Beads (0.5 mm dia)
10X DNase 1 Buffer
DNase 1
DNase Inactivation Reagent
–20°C
–20°C
–20°C
–20°C
* If a precipitate develops during storage, dissolve the precipitate before use by warming
the solution to 37°C.
† Phenol:Chloroform:IAA is a poison and an irritant. Contact with it will cause burns.
Use gloves and other
personal protection equipment when working with this
reagent.
‡ This reagent contains guanidinium thiocyanate, a potentially hazardous substance; use
with appropriate
caution.
Vortex Mixer w/ adapter (Cat Ambion #AM10024)
95ºC Heating Blocks filled with deionized water
30ºC & 37ºC Water Bath Incubators
Swinging bucket centrifuge @ 4ºC (G-309)
1.2% Agarose Gels
DNA Gel boxes
10X Nucleic Acid Sample loading dye
HyperLadder I Wide Range Markers
Gel Running Bufffer (1 liter electrophoresis buffer 1x TBE)
Ethidium Bromide
Power Supply
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NOTES:
Lab bench and pipettors
Before working with RNA, it is always a good idea to clean the lab bench and
pipettors with an RNase decontamination solution (e.g. Ambion RNase Zap Solution).
Gloves and RNase-free technique
• Wear laboratory powder free gloves at all times during this procedure and change
them frequently. They will protect you from the reagents, and they will protect the
RNA from nucleases that are present on skin. • Use RNase-free tips to handle the
wash solutions and the Elution Solution, and NEVER put used tips into the kit
reagents.
Keep reagents on ice
Always close pipet tip boxes
Dispose of tips in waste containers, not on the bench
Never use tips more than once!
B. 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 the media is exhausted. Growth of the yeast cultures is 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 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
which 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
< 107
1-5 x 107
5 x107 - 2 x 108
2 x 108
Haploid yeast cells contain approximately 1.2 pg of RNA per cell.
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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 because glucose was depleted over the time course.
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 (ie using Lyticase).
1) Each group receives a yeast cell pellet 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. Samples were standardized to contain 21 OD600 units. (i.e. 30 ml x 0.7
OD600 Absorbance units) Predict approximately how much RNA you should isolate.
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 (large) eppendorf tube. Transfer the cells from the 15 ml conical
tubes to the eppendorf tube.
6) Place the eppendorf tubes in the microcentrifuge (balancing tubes). Spin at 11,200g for
3 min @ 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 your tube to hood.
Add
3µl ß-mercaptoethanol (a reducing agent that smells very bad)
320 µl of lyticase (enzyme that degrades cell wall components)
to the resuspended pellet.
9) Place tubes in the incubator 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
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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
ß-mercaptoethanol.
10) Spin in microcentrifuge at 5600 g for 2 min. Remove supernatant by pipeting (discard
the supernatant in hood to contain the BME smell).
11) Add 500µl potassium phosphate/sorbitol buffer to the pellet and resuspend. Repeat
step 10 (one time).
12) While centrifuging make sure your ice bucket contains:
1 tube (screw cap) containing glass (zirconia) beads
1 tube of Lysis Buffer
1 tube of 10% SDS (Warm @ 37C to dissolve any precipitate)
1 tube of Phenol:Chloroform:IAA
Clean RNA isolation area with 'RNAse ZAP' solution
(1 spray bottle for lab)
Isolating RNA
The RiboPure-Yeast method disrupts yeast cell walls by beating cells mixed with an aqueous lysis buffer,
SDS, phenol and 0.5 mm Zirconia Beads on a vortex adaptor (e.g. Ambion Cat #AM10024) for 10 min. Up
to 3 x 108 cells can be processed at a time. The lysate is centrifuged to separate the aqueous phase, which
contains the RNA, from the lower organic phase, which contains proteins, polysaccharides, and other
cellular debris. The aqueous lysate is diluted with Binding Buffer and ethanol, and is drawn through a
glass-fiber filter, which immobilizes the RNA. Contaminants are washed from the filter, and finally the
RNA is eluted in a low ionic strength solution. Residual DNA is removed by treating the RNA with
Ambion DNAfree ™ reagents (DNAse enzymes), included in the kit. As with all glass-fiber filter
purification methods, 5S ribosomal RNAs and tRNAs are not quantitatively recovered using the RiboPureYeast Kit. The entire RNA isolation procedure and DNase treatment require approximately 1.5 hrs, starting
with fresh, snap-frozen cultured yeast cells. The resulting RNA should be of superb quality, free of DNA
and proteins, and suitable for use in virtually any downstream application.
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")
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C. Cell Disruption and Initial RNA Purification
1. Resuspend cells in lysis reagents
Add the following to your yeast cell pellet in the order shown, and resuspend by
vortexing vigorously for 10–15 seconds:
(CAUTION: these reagent are extremely caustic; handle with respect)
Amount Component
480 µL Lysis Buffer
48 µL 10% SDS (Check to make sure no precipitate is present)
480 µL Phenol:Chloroform:IAA
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2. Add mixture to Zirconia Beads
Transfer the mixture of cells and lysis reagents to the prepared tube containing ~750 µL
cold Zirconia Beads, and securely tighten the cap.
3. Beat cells for 10 min on vortex mixer with a vortex adapter
Position the sample tubes horizontally on the vortex adapter with the tube caps towards
the center. Turn the vortex mixer on at maximum speed and beat for 10 min to lyse the
yeast cells.
4. Centrifuge the lysed cells, then transfer the aqueous phase to the 15 mL tube that
contains 1.9 ml of Binding Buffer as explained below.
a. Centrifuge for 5 min at 16,100 g at room temp to separate the aqueous phase,
containing the RNA, from the organic phase.
b. Transfer the aqueous phase (top), containing the partially purified RNA, to the
15 mL tube labeled “Binding Buffer”. Typically, the recovered volume from the cell lysis
will be approximately 530 µL.
D. Final RNA Purification
1. Before you start:
Check Wash Solution 1 and redissolve by heating to 37°C if necessary
A precipitate may form in the Wash Solution 1. Examine the reagents carefully before
use, and if a precipitate is visible, redissolve it by heating the solution(s) to 37°C, and
agitating the tube occasionally.
Preheat Elution Solution to ~95°C
Place 50 µL Elution Solution per sample into the heat block set to 95–100°C. The
preheated Elution Solution will be used in step D.8.
Inspect the Filter Cartridges
Briefly inspect your Filter Cartridge before use. Occasionally, the glass fiber filters may
become dislodged during shipping. If this is the case, gently push the filter down to the
bottom of the cartridge using the wide end of a RNase-free pipette tip.
NOTE:
All centrifugation steps should be done at the maximum speed setting on a microfuge.
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2. Your sample now has a volume of ~2.4 ml (~530µL recovered from the lysis step and
1.9 ml of Binding Buffer.) This corresponds to 350 µL Binding Buffer for each 100 µL of
partially purified RNA.
3. Add 100% ethanol to your sample
Add 1.25 mL 100% ethanol and mix thoroughly. (This corresponds to 235 µL ethanol for
each 100 µL of partially purified RNA.)
4. Draw sample through a Filter Cartridge
a. Apply 700 µL of the sample mixture to a Filter Cartridge assembled in a Collection
Tube. The total volume of the lysate/ethanol mixture is ~3.7 mL, since the Filter
Cartridge can only accommodate 700 µL, you will need to draw the lysate mixture
through the Filter Cartridge in several applications of 700 µL at a time.
b. Centrifuge for ~0.5–1 min or until the lysate/ethanol mixture is through the filter.
c. Discard the flow-through and return the Filter Cartridge to the Collection Tube.
d. Repeat as necessary to pass the entire sample through the Filter Cartridge.
NOTE:
The RNA is now bound to the filter in the Filter Cartridge.
5. Wash Filter Cartridge
a. Wash the filter by adding 700 µL Wash Solution 1 to the Filter Cartridge, and
centrifuging for ~1 min or until all of the liquid is through the filter.
b. Discard the flow-through and return the Filter Cartridge to the same Collection Tube.
6. Wash Filter Cartridge with Wash Solution 2/3
a. Wash the filter by adding 500 µL Wash Solution 2/3 to the Filter Cartridge. Draw it
through the filter as in the previous step.
b. Repeat with a second 500 µL aliquot of Wash Solution 2/3.
7. Centrifuge for to remove excess wash solution from the filter
a. Centrifuge the Filter Cartridge for 1 min to remove excess wash.
b. Transfer the Filter Cartridge to a fresh 2 mL Collection Tube.
8. Elute RNA in 2x25 µL preheated Elution Solution
a. Elute RNA by applying 25 µL Elution Solution, preheated to 95–100°C, to the center
of the filter.
b. Centrifuge for 1 min. DO NOT DISCARD YOUR RNA!
c. Repeat the elution step with a second 25 µL aliquot of preheated Elution Solution into
the same Collection Tube. Make sure your RNA is clearly labeled with your name, date,
sample info etc.
E. DNase I Treatment
RNA isolated using RiboPure-Yeast will contain contaminating chromosomal DNA. To
remove it, we recommend including this DNase 1 treatment. The DNase Treatment and
Removal Reagents provided with the RiboPure-Yeast Kit include ultrapure DNase I and
reaction buffer that are rigorously tested to be sure that no RNase is present. This
procedure makes use of a simple process to inactivate the DNase without the risk
incurred by heating the RNA, or the hassles of adding EDTA or extracting with
phenol/chloroform.
1. Assemble the DNase digestion reaction
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Assemble the DNase digestion reaction at room temp.
Amount
50 µL
5 µ (1/10th vol)
4 µL
Component
RNA sample (from step D.8)
10X DNase 1 Buffer (i.e. 5–10 µL)
DNase 1 (8 UNITS)
2. Incubate 30 min at 37°C
Incubate the DNase digestion reaction for 30 min at 37°C.
3. Treat with 0.1 volume DNase Inactivation Reagent
a. Resuspend the DNase Inactivation Reagent by flicking or vortexing the tube, then add
0.1 volume DNase Inactivation Reagent to your sample. For a typical 59 µL DNase
digestion reaction, use 6 µL DNase Inactivation Reagent.
b. Mix by vortexing and allow reactions to stand 5 min at room temp.
c. Centrifuge 2–3 min at top speed in a microfuge (≥10,000 x g) at room temp to pellet
the DNase Inactivation Reagent. Transfer the RNA (supernatant) to a fresh 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?)
F. RNA Storage
RNA samples can be stored at –20°C for up to 1–2 months, or at –80°C for longer than 2
months.
This a good stopping point, so we will freeze our samples until next week to finished the
purification and analysis.
STOP and Store samples at -70 ºC
Continue here at the next lab.
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.
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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
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?)
1 = Degraded RNA
2 = Good RNA
3 = Good RNA