Unit 1: week 4
Yeast Mating “The Effect” Part IIIa
Blackboard Assignment due by Monday 9:59 PM
Reading:
MBoC4 (the text):302-305 From DNA to RNA to end of page.
‘basic’ PCR method: http://www.dnalc.org/shockwave/pcranwhole.html
RT-PCR animation:
http://www.bio.davidson.edu/courses/Immunology/Flash/RT_PCR.html
=====
This week's lab looks incredibly long. That is mainly because each step is written out and
the manual includes a lot of background material. Make sure to read over the protocols
and have any technical (or philosophical) questions ready. You need to know both what
you are doing in each step and why you need to do it.
In this lab you will begin to characterize the intra-cellular changes that occur in a mating
type a cell when exposed to the mating pheromone from an  cell. That is, you will look
at how starting the mating response effects the transcription of certain genes.
In most every ‘cell-level’ biology class you have had you hear how DNA is transcribed
into RNA and RNA is translated into proteins (the ‘Central Dogma’). Most of the time
you look at DNA (like genotyping in Bio111) or proteins (the visible phenotypes,
Western blot analysis in Genetics, Molecular or Developmental). In this class you will
look at RNA. By looking at the relative amount of transcripts (RNA) we will be able to
see which new genes does the cell need to use in order to shmoo and fuse or tell which
other genes it needs to turn down or off so that the cell cycle stops. This time we will be
performing the comparison between wildtype yeast that are budding and wildtype yeast
that are shmooing. In the second half of the semester you will decide how to best apply
this procedure to examine your mutant yeast that can’t mate (so keep that in mind).
Comparative RT-PCR is a modified version of RT-PCR—but what is that?
The following diagram (from the Ambion.com website) provides an overview of the
whole process and the next few paragraphs provide names, definitions and explanations.
Before starting that I would like to recognize that Ambion, Inc. graciously donated the
Comparative RT-PCR kits we will use as well as some of the RNAse inhibiting
precautions.
During the first week we are isolating ‘RNA population I’ and ‘RNA population II’
RNA from yeast that are budding and RNA from yeast undergoing the mating reaction
(induced with  factor). In the second week you will use specially tagged primers to
make cDNA from your RNA samples (Reverse Transcription with the tag10 and tag50
primers). You will then use that cDNA in PCR reactions that are designed to determine
differential gene expression and detect the differences in expression by running an
agarose gel. (Differential gene expression simply means that different genes are
transcribed in different tissues or under different growth conditions.)
Cell Bio Manual 2003
16
Figure 1: Overview of Comparative RT-PCR: From Ambion Intraspec Comparative RT-PCR protocol:
Figure 2. pdf of the entire protocol is linked from http://www.ambion.com/catalog/CatNum.php?1742
But what is RT-PCR?
Reverse transcription (RT). Since transcription is the process of reading a DNA
template to create a strand of RNA then reverse transcription is the process of using an
RNA template to create a strand of DNA called complementary DNA (cDNA).
Why would you want to reverse transcribe?
1) DNA (even single stranded DNA) is more stable than RNA so you are converting
your sample into a form is less likely to fall apart in the middle of your experiment.
2) DNA represents the entire genome (pieces of DNA the cell is using and regions or
genes it is not). RNA represents only the parts of the genome that the cell is actively
transcribing under your experiments growth conditions. Therefore cDNA gives you a
Cell Bio Manual 2003
17
pool of relatively stable molecules that represent all the genes that the cell needed to
shmoo vs all it needed to bud (for example).
3) RNA levels and therefore cDNA levels are a measure of the relative abundance of
the encoded protein. For example a cell might only need one copy of the RNA encoding
a rare receptor—not many copies of that protein are needed so not many RNA templates
are made. However the cell contains millions of actin monomers therefore more copies of
actin RNA are transcribed so that more copies of the actin protein can be made. In the
average cell some genes are used more. We can determine this difference by looking at
the relative amounts of actin cDNA vs rare receptor cDNA.
In our case we could also look at whether a cell makes more of the receptor when it is
budding or when it is shmooing.
4) In Comparative RT-PCR, the RT reaction adds a ‘tag’ to the cDNA so that you
can tell your ‘+factor’ samples from your ‘-factor’ samples just by the size of the
reaction product. Our cDNA is created by using an RT primer that includes a primer
binding site (PBS), a linker region and a string of thymines (abbreviated ‘dT’). What do
you think the string of T’s hybridizes with?
The PBS and linker regions features are specific to our set of reactions and not part of the
standard RT reaction. By using the Tag10 primer for one condition (+alpha) and the
Tag50 primer a different condition (-alpha) we end up with two pools of cDNA that can
be distinguished by the relative size of the DNA product.
What is PCR?
PCR= polymerase chain reaction This is the equivalent of DNA replication in a
microfuge tube. You have encountered PCR before in Bio111 (when you determined
your genotype) and so this should be a review. Briefly, two short pieces of DNA are used
as primers to start off the copying of a DNA template. In our case one the primers will
hybridize with the PBS on the 3’ end of the cDNA. Because every cDNA has this site this
will not help us identify a particular gene. That is the job of the second primer. The
second primer is specific for a particular gene, for example, Tubulin (TUB2), Ste2, Kar1,
or Far1. Each of these is a gene that known to be involved in the cell cycle or the
mechanism of mating.
How will we detect differences? (Where is the comparison in comparative RT-PCR?)
We will detect differences in gene expression by setting up the PCR reactions so that they
contain PBS primers, a gene specific primer and exactly equal amounts of cDNA from
out two different pools. During PCR the cDNAs from the tag10 and tag50 pools will act
Cell Bio Manual 2003
18
as ‘competitors’ for the pool of primers. After the reaction is complete the products are
separated by agarose gel. Each lane should only contain two bands. The higher MW band
represents the copies from the cDNAs tagged with tag50. The lower MW band represents
the copies from the cDNAs tagged with tag10. The relative intensities of these bands
indicate differences in gene expression.
________________________________________________________________________
Now that that is clear—Let’s isolate RNA
Isolation of total RNA from yeast
In order to further characterize the mating reaction you will be isolating total RNA from
TWO cultures of DLY1. One culture contains cells in logarithmic growth phase grown in
YEPD. The other contains 'log' cells, in YEPD, that have been treated for over 3 hours
with  factor. (so what should they be doing?…) Next week you will begin using a
procedure called Comparative RT-PCR to determine the effect of  factor on
transcription of a gene involved in the mating reaction, and TUB2, the gene encoding
tubulin (a cytoskeletal protein)
One of the biggest problems in most experiments is being able to tell the difference
between a 'result' and an artifact of the experiment. Previously we used growth on
different types of media as positive and negative controls to detect complementation and,
therefore, mating. In these experiments we will be detecting an mRNA that is
'constitutively' expressed as a positive control to show that the methods are working.
(constitutive means ‘essential’ so biologically speaking a gene that is constitutively
expressed is always expressed) Our positive control is the TUB2 gene—the gene that
encodes the cytoskeletal protein tubulin. As you prepare the RNA consider what you will
use for a negative control, what it will show, why it is appropriate for this experiment.
RNA isolation is tricky. You must get rid of RNases
Your objective is to isolate a large quantity (perhaps 50 whole micrograms) of
undegraded total RNA. Read all instructions before even 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 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 afternoon, experimentally speaking. The samples
you isolate today will be the basis for the blot you perform this week and next week AND
will be saved so that you can use them as controls in your experiments that characterize
you mutants later in the semester. Extra caution now will save you a bunch of work then.
A 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
Cell Bio Manual 2003
19
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 Erase' or ‘RNA away’ (special
detergents that helps a little). 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.
Preparing yeast cell lysate
Yeast contain a hard cell wall that help them survive as single cells in a harsh
environment. In order to get to the RNA in the cytoplasm we must break through this cell
wall—we must lyse the cells.
1) Each group receives 20mls of non-induced DLY1 cells and 20mls of  factor induced
DLY1 cells ( factor was added to 1ug/ml of media approximately 3 hours before
class) in 50ml conical tubes. All cultures are in log phase growth.
2) Label the non-induced DLY1 tube "N" and the induced cell tube "I" also add a
distinguishing mark so you can identify your tube from other groups.
3) Place N and I tubes in centrifuge (balancing it). Centrifuge at 5000rpm for 5min to
pellet the cells. There will be more than one group using the large centrifuge in the
adjoining prep room, be good 'lab citizens' and share the centrifuge.
4) While spinning make sure know where the following supplies are:
yellow and blue tips for pipetmen (boxes)
Two 2ml screwcap tubes containing 300ul of 4.5mm glass beads
Phosphate buffered saline
Ice bucket (with ice)
2x Tissue and Culture Lysis Buffer (2x T&C Lysis)
MPC protein precipitation buffer
5) Remove tubes from centrifuge. Carefully pour supernatant into waste container. Try
not to disturb pellet. Add 250ul of Phos.Buff.saline. Resuspend the pellet by pipetting
with a p1000.
6) Label 2ml screwcap ‘bead’ tubes. Transfer the cells from the conical tubes to the
appropriately labeled bead tube. Tap on the sides and invert to mix cells with glass
beads. Place on ice.
7) Add 300ul 2X Tissue and Culture Lysis buffer. Invert to mix with beads and cells.
(why did the T&C Lysis buffer have to be supplied in a ‘2x’ stock? What does the 2x
mean?)
Isolating RNA
FROM THIS POINT ON
DO NOT TOUCH ANYTHING ON YOUR BENCH WITHOUT WEARING GLOVES.
DO NOT 'DOUBLE TOUCH' TIPS
DO NOT WALK AROUND WITH OPEN TUBES
KEEP SAMPLE ON ICE UNLESS SPECIFICALLY INDICATED OTHERWISE
DO NOT LAUGH, TALK, OR BREATHE OVER OPEN TUBES
(Sometimes paranoia is justified—the RNases are out to get you)
Cell Bio Manual 2003
20
8) Using the ‘Bead Beater’ (side bench) shake the cells at 4800rpm for 2.5min. Place tube
on ice. (Bead Beater only hold one tube—again be good lab citizens)
2X Tissue and Culture Lysis buffers and chaotropes that will help stabilize the RNA in
solution
This step breaks open the yeast by brute force. Why do you think the glass beads are
included? Why must samples be placed on after ‘bead beating’?
9) After both samples have been ‘beaten’ place on ice for 2min.
10) Centrifuge at 50% (7000g) for 3 min to break foam.
11) While tubes are spinning- label clean microfuge tubes "N" or "I"
12) Transfer supernatant to clean tube. To do this place yellow tip all the way through
beads and move as much of liquid as possible. You should be able to remove at least
250ul.
13) Add 1ul of 50ug/ul proteinase K to each tube (get from me), vortex for 3sec then
incubate at 65C (heating block on side bench) for 15min. Every 5min vortex again.
By the name ‘proteinase K’ what do you think this enzyme is doing? Why is this
important in our quest for high quality RNA? Why vortex every 5 min?
14) Place samples on ice 3min
15) Add 150ul MPC protein precipitation buffer
16) Vortex for 10sec then incubate on ice for 3min
17) Centrifuge at 100% (14000g) for 10min
While sample is spinning label two clean tubes.
18) Place sample on ice 1min. You should see a significant, white, compact pellet. (If you
don’t see it, come get me). Carefully move each supernatant into a separate clean tube.
19) Add 550ul Isopropanol (2-propyl alcohol, found in hood—bring your samples to it,
do not remove it from the hood)
20) Mix the sample by inverting 40 times.
21) Centrifuge in REFRIGERATED centrifuge 14,000g x 10min. We only have one
refrigerated microfuge and I need to show you how to operate it. Keep all samples on
ice, talk to me so groups can coordinate their spins.
22) Pour off supernatant. Pulse spin (10 sec, 100%, your microfuge). Carefully remove
any residual liquid without disturbing the pellet. (It will be a small, relatively clear,
smudge near the bottom.)
THIS IS YOUR RNA--place the tube on ice –
At this point the RNA still has traces of genomic DNA in it. All trace DNA must be
removed. (Think about the protocol you will be performing. Why/How could trace
genomic DNA radically throw off your results?)
Your RNA should always be stored on ice or frozen to slow degradation (why would
cold slow degradation?). Even if ‘only’ moving it from place to place or removing
aliquots keep the RNA on ice.
23) Add 50ul of ‘Tris-cation buffer’ (side bench) to each pellet and tap on the tube to
help resuspend the pellet in the buffer.
Question: What types of RNA have you just isolated? (how many different kinds of
RNA are in a cell?) What other cellular molecules could still be in this sample (think
about chemically similar compounds)? How can RNA analysis help you characterize
Cell Bio Manual 2003
21
your 'unknown' strain later in the year? What will it tell you about where your strain is
blocked in the mating reaction?
Next week you will be reverse transcribing your RNA and then setting up PCR. You just
isolated RNAs but a DNA prep would have been very similar. In fact, in all likelihood
your RNA prep contains trace amounts of genomic DNA (gDNA). In order for our PCR
to be ‘comparative’ every single piece of gDNA must be removed from the prep (Why is
this so critical? What harm could it be if there was just a handful of DNA among the
thousands of RNAs in your prep?) The next step ensures that DNA will not be a part of
your RNA preps.
24) Take your samples, on ice to the side bench and add 1ul of DNase to each.
(It ends in ‘-ase’, what does DNase do and why will it help prepare your sample for RTPCR?)
25) Place your samples at 37C (hot block) for 25min.
While incubating make sure that you are prepared for the quantification steps, add notes
to your notebook so that you would
a) be able to tell someone what you actually did (not just what was written) and
b) be able to repeat this process even more successfully – add helpful hints.
26) Vortex the ‘DNase Inactivation Reagent: Ambion’ to form a slurry. Add 5ul of this
slurry to each of your samples.
NOTE: The important reagent is the white part of the slurry. This part will inactivate the DNase
and remove divalent cations (Mg++ and Ca++) from the sample. Look at the reagent when it is in
the pipet tip—it should be primarily white, not clear.
Thinking about what we will be doing with these samples, why is it important to remove
the DNase? Why remove the divalent cations?
27) Flick the samples to mix, incubate 2min at room temperature, and then flick again.
28) Centrifuge samples at 75% (10,000g) for 1min to pellet the DNase Inactivation
Reagent.
YOUR DNA-free RNA sample is the supernatant.
When performing any further experiments be sure to avoid the white pellet at the bottom.
Quantifying RNA
When performing the reverse transcription part of RT-PCR you need to know how much
RNA you have. We will use spectrophotometry to determine how much RNA you have
as well as how ‘clean’ it is (how free of protein contaminants).
1) Make sure that spectrophotometer is on (located in the adjoining prep room research
lab). The UV lamp must be turned on and must have 5min to warm up.
The life of this lamp is GREATLY reduced if you keep turning it on and off. When quantifying
RNA coordinate with other groups and leave a note on the spectrophotometer saying when it was
turned off. It must have a chance to cool for at least 45min before being turned on again. (so
today leave it on so your lab mates can get to go to dinner)
2) Get 2 plastic UV ultramicrocuvettes from the drawer below the spectrophotometer.
Hold cuvettes by the edges--fingerprints on the flat surface will cause inaccurate readings.
Cell Bio Manual 2003
22
3) Place 195ul of sterile distilled water into each cuvette. Add 5ul of your uninduced
yeast RNA into one. Add 5ul of your induced yeast RNA into the other. Be careful to
avoid the white pellet.
What dilution of RNA did you just perform? 5 in 200 equals a one in ________dilution.
This means that you diluted the sample by a factor of ________ (hint: it’s the same # as
above).
4) Set up Spectrophotometer to read absorption at 260nm and 280nm
Take a reading of the blank (Distilled water + 5ul buffer) before each sample.
Uninduced Blank
Induced Blank
OD260
OD280
(Make sure to leave the blank by the spectrophotometer for other groups, wash out your
samples’ cuvettes 5 times with dH20, then with EtOH, leave upside down on kimwipe by
the spec. to dry)
5) Calculate the 280/260 ratio (using readings that have been corrected for the blank). A
'very clean' sample will have a 280/260 ratio of between 0 approximately 0.4 to 0.55
INDUCED 280/260=
UNINDUCED 280/260=
6) Calculate the concentration of RNA in each sample using the following equation.
(Note that the equation only uses the OD260.)
[ug/ml] = (OD 260) * (Dilution factor)* 40
bold= conversion coefficient for single stranded RNA
italics= you calculated the dilution factor in #3
INDUCED SAMPLE CONCENTRATION:
UNINDUCED SAMPLE CONCENTRATION:
AFTER you have calculated the sample concentration add 1ul of Super RNAsIn to each
sample. RNAsin inhibits RNAses (and keeps your 'RNAs…in…'). This compound should
help keep your samples intact.
7) Make sure that your RNA tube is well labeled—label should say what it is, its
concentration and a distinguishing mark so you can retrieve it.
8) Place your RNA in the rack in the ‘live fridge’ freezer.
9) Clean up your lab bench. Dispose of all waste properly in containers in the hood.
Before leaving record the RNA concentration and purity of your samples on the board.
Cell Bio Manual 2003
23