RT-PCR: Detecting Gene Expression
You have a gene you are interested in, and the gene may cause the kidneys to
develop. Thus, you want to see if the gene is “on” at the time that kidneys develop in
embryonic mice.
If the gene is “on” the gene will be coding for mRNA (messenger RNA; carries
the code for a protein from DNA to the ribosome in the cytoplasm), and the protein coded
by that mRNA will be made.
However, it is not always possible to record whether the protein is being made
due to lack of a specific antibody for the protein from that gene.
Another way to analyze for gene expression (whether the gene is on and making
mRNA) is to look for the presence of specific mRNA from that gene. mRNA analysis is
typically done by the techniques of RT-PCR, qRT-PCR (see below), or Northern blot
analysis.
We will use RT-PCR (reverse transcription-polymerase chain reaction) to detect
RNA expression of the gene for phospholipase C-gamma.
The Protocol (see figure immediately below; taken from our Textbook World of the
Cell by Becker et al.):
First, we will isolate total RNA from oocytes. Total RNA has mRNA, rRNA, and
tRNA (ribosomal and transfer RNA, respectively).
Second, Then we will add Reverse Transcriptase (RT) – which is an enzyme first
found in retroviruses- that will take the mRNA and make it into complimentary DNA
(cDNA). This is the reverse of transcription (making mRNA from DNA). RNA is
reverse transcribed to a single stranded
complimentary DNA (cDNA) molecule.
Retroviruses are viruses with RNA and no
DNA; the virus contains the RT enzyme that
makes DNA from the viral RNA (in the host
mammalian cell). The RT step takes place in
the beginning of the PCR in the PCR
machine.
Third, we will use a PCR machine to
make many many copies of the cDNA. We
will obtain copies of part of the gene for a
certain protein called Phopholipase C-
(PLC-gamma). Thus, we are seeing if the
PLC-gamma gene is on in Xenopus oocytes.
The PCR reaction can use an oligo dT
primer to synthesize the first strand by
exploiting the presence of a poly(A) tail on
mRNA. The use of an oligo dT prime to
initiate first strand synthesis allows
production of a cDNA from all mRNA
transcripts (see Figure 20-31 from our textBecker’s World of the Cell).
1
This is how a cDNA library is
constructed. But the use of oligo d(T)
primers is not necessary in most RT-PCR
reactions and can decrease the specificity
of the PCR reaction. We will use a 3’ gene
specific primer for the first strand
synthesis. Once the cDNA strand is made,
two gene specific primers (GSP) are used
to amplify the gene or part of the gene of
interest. The primers used for PCR can
flank just a small region of the mRNA, or
can be designed to amplify the entire
mRNA molecule. Amplification of an
entire mRNA can be challenging if the
mRNA is greater than 3.5 kb.
PCR makes many copies of a
fragment of DNA (i.e., gene) and is a
molecµlar technique based on the concepts
of DNA replication. The components of a
PCR reaction are
(1) DNA polymerase (makes
DNA),
(2) dioxynucleotides (dNTPsbuilding blocks of DNA),
(3) primers for replication of a
specific region of DNA by DNA
polymerase (DNA polymerase actually
requires short strands RNA that are called
primers- the two different primers only
bind at ends of a segment of DNA within
the gene of interest- the PLC-gamma geneso that you make many copies of the bit of
DNA located within the primer binding
sites), and
(4) the proper buffer components
for the polymerase to be active.
As you recall from
lecture/textbook, DNA replication involves
steps of (1) unwinding the DNA double
helix, (2) the laying down RNA primers to
initiate DNA replication, and (3) DNA replication or elongation. PCR is based on this
same principle.
There are three major steps in PCR (1) denaturation (high temperature breaks the
weak bonds binding the two strands of DNA together, making the double stranded DNA
into single stranded DNA) (2) annealing (raising the temperature to allow DNA primers
to bind the specific DNA sequence they are complimentary to- forming Watson-Crick
2
base pairs), and (3) elongation (a low temperature, DNA replication with DNA
polymerase) (see Figure 19A-1—note the suggested temperatures).
Denaturation of the DNA molecule is carried out at 95 C. This temperature will
denature all human enzymes, and therefore a thermal-stable DNA polymerase is used
(thermal-stable means the enzyme can withstand this high temperature—like
ribonuclease that we studied; why is ribonuclease unusual??). We will use Taq DNA
polymerase, cloned from the bacterium Thermus aquaticus.
An important control for RT-PCR is a “no RT control.” RT-PCR is designed to amplify
a specific nucleic acid sequence from RNA. In the process of isolating RNA, genomic
DNA can contaminate the RNA prep. This contamination can lead to false positive
results when looking for gene expression. There are a few methods to avoid false
positives (1) use DNase to remove the DNA from the RNA prep, (2) design primers that
flank an intron, and (3) use a no RT control reaction every time you do RT-PCR.
In this lab you will perform RT PCR using RNA isolated from fresh Xenopus oocytes.
You will amplify a region of the Phospholipase C- gene. The RNA samples will be
isolated from fresh Xenopus oocytes. The RT-PCR amplification products should be 678
nucleotides (nt) long for phospholipase C-.
Primers to use:
5’PLC-gamma
3’PLC-gamma
TGTGGMGNGGIGAYTAYGG
ATATGAATTCTGGTGGMGNGGIGAYTAYGG
The PLCG (and PLD) primers are considered “degenerate” which means that the primer
stock has a mixture of oligos representing multiple codon possibilities. Degenerate
primers are used when exact nucleotide sequence is not known. This is common when
we know the amino acid sequence of a protein (and/or going from protein sequence of
one organism to another). For example, we know that an amino acid sequence has a
particular amino acid, but this amino acid could be coded by different combinations of 3
nucleotides. The degenerate code usage is below.
M = A or C
R = A or G
W = A or T
S = C or G
Y = C or T
K = G or T
H = A or C or T
V = A or C or G
B = C or G or T
D = A or G or T
N = A or C or G or T
I = inosine
We might also look at another enzyme; phospholipase D (PLD) and use these primers:
5' PLD
TGGGCICAYCAYGARAA
3' PLD
TCRTGCCAIGGCATICKIGG
3
The following procedures will be preformed:
Part I) First lab period:
a. high quality RNA will be isolated from Xenopus oocytes
b. quantification of RNA by the spectrophotometer
c. RT-PCR reaction
Part II) Second lab period:
a. gel electrophoresis to analyze the RT-PCR products
Part I: Oocyte RNA with Stratagene’s “Absolutely RNA Miniprep Kit”
The objective of this part of the experiment is to isolate high quality RNA, which
means RNA that is not degraded and is free from DNA contamination.
The Stratagene kit uses a spin column packed with a silica-based matrix that
specifically binds RNA in the presence of the chaotropic salt guanidine thiocyanate.
"Chaotropic" means chaos-forming, a term which in biochemistry, usually refers to a
compound's ability to disrupt the regular hydrogen bond structures in water. This
hydrogen bonding profoundly affects the secondary structure of polymers such as DNA,
RNA and proteins as well as how water soluble something is. Chaotropic salts increase
the solubility of nonpolar substances in water. The chaotropic salts are used to alter the
characteristics of the water molecules which surround the DNA molecule. In this way the
positive H cloud is weakened so that the DNA molecule can more easily bind the the Sibased matrix (http://www.protocol-online.org/archive/posts/8364.html).
Chaotropic salts also denature proteins because they have the ability to disrupt
hydrophobic interactions. They do not denature DNA or RNA but the denaturation of
proteins not only aids in removing the proteins from the RNA but also prevents RNases
from degrading the RNA molecules. Thus, they have two functions in the Kit: (1)
denature cellular proteins (such as DNAse and RNAse), and (2) the high concentration of
salt also facilitates binding of the nucleic acids DNA and RNA to the silica membrane in
the column (http://www.clontech.com/clontech/techinfo/faqs/mn.shtml).
A series of washes removes contaminants from the RNA and then the RNA is
eluted off the column. This procedure eliminates the need for toxic phenol-chloroform
extractions and time-consuming ethanol precipitations. The washing buffer contains nonchaotropic salts and 70% ethanol. Ethanol precipitates nucleic acid and this attaches the
RNA more firmly to the matrix in the spin cup.
Subsequently, washing is needed to get rid of the chaotropic salts cause these
things will inhibit just about anything (PCR,qPCR) and even the new generation of ABI
sequencers can't always handle these kits (http://www.protocolonline.org/archive/posts/8364.html).
We use different washes: high salt and low salt. The high salt decreases the
negative charge on the RNA allowing stronger interactions with the spin cut matrix. The
low salt increases the repulsion between the RNA and the cup matrix, allowing elution of
the RNA from the spin cup (http://www.protocol-online.org/archive/posts/8364.html).
The protocol incorporates a DNase digestion step to aid in removing any
remaining DNA.
Material provided
Quantitya
Storage conditions
Lysis Buffer
35 ml
Room temperature
4
β-Mercaptoethanol (β-ME) (14.2 M) 0.3 ml
Room temperatureb
RNase-free DNase I (lyophilized) 2600 U
Room temperaturec
DNase Reconstitution Buffer
0.3 ml
Room temperature
DNase Digestion Buffer
2 × 1.5 ml
Room temperature
High-Salt Wash Buffer (1.67×)
24 ml
Room temperature
Low-Salt Wash Buffer (5×)
17 ml
Room temperature
Elution Bufferd
12 ml
Room temperature
Prefilter Spin Cups (blue) and 2-ml receptacle tubes
50
Room temperature
RNA Binding Spin Cups and 2-ml receptacle tubes
50
Room temperature
1.5-ml microcentrifuge tubes
50
Room temperature
--------------------------------------------------------------a Sufficient reagents are provided to isolate total RNA from 50 samples of 40 mg
of tissue or 1 × 107 cells.
b Once opened, store at 4°C.
c Once reconstituted, store at –20°C.
d 10 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA.
Other items needed:
Diethylpyrocarbonate (DEPC)- inhibits RNase
Ethanol [100% and 70% (v/v)]- to precipitate RNA or DNA
Homogenizer
Spectrophotometer set to 260 nm
Note: any dangerous chemicals?? The Material Safety Data Sheet (MSDS) information for
Stratagene products is provided on Stratagene’s website at http://www.stratagene.com/MSDS/.
Simply enter the catalog number to retrieve any associated MSDS’s in a print-ready format.
Preventing RNase Contamination
Ribonucleases are very stable enzymes that hydrolyze RNA- remember we noted this in
an earlier lecture (why are they unusual?). RNase A can be temporarily denatured under extreme
conditions, but it readily renatures (without chaperones- typical proteins irreversibly precipitate
when their nonpolar amino acids are revealed during unraveling). RNase A can therefore survive
autoclaving and other standard methods of protein inactivation. The following precautions can
prevent RNase contamination:
♦ Wear gloves at all times during the procedures and while handling materials and
equipment, as RNases are present in the oils of the skin.
♦ Exercise care to ensure that all equipment (e.g., the homogenizer, centrifuge tubes, etc.)
is as free as possible from contaminating RNases. Avoid using equipment or areas that have been
exposed to RNases. Use sterile tubes and micropipet tips only.
♦ Micropipettor bores can be a source of RNase contamination, since material accidentally
drawn into the pipet or produced by gasket abrasion can fall into RNA solutions during pipetting.
Clean micropipettors according to the manufacturer's recommendations. Stratagene recommends
rinsing both the interior and exterior of the micropipet shaft with ethanol or methanol.
Disposable sterile plasticware is generally free of RNases. If disposable sterile
plasticware is unavailable, components such as 1.5-ml microcentrifuge tubes can be sterilized and
5
treated with diethylpyrocarbonate (DEPC), which chemically modifies and inactivates enzymes
(refer to Sambrook, et al.).
Treating Solutions with DEPC
Treat water and solutions (except those containing Tris base) with 0.1% (v/v) DEPC in distilled
water. During preparation, mix the 0.1% DEPC solution thoroughly, allow it to incubate
overnight at room temperature, and then autoclave it prior to use. If a solution contains Tris base,
prepare the solution with autoclaved DEPC-treated water.
Caution DEPC is toxic and extremely reactive. Always use DEPC in a fume hood. Read and
follow the manufacturer's safety instructions.
Nondisposable Plasticware
Remove RNases from nondisposable plasticware with a chloroform rinse. Before using the
plasticware, allow the chloroform to evaporate in a hood or rinse the plasticware with DEPCtreated water.
Glassware or Metal
To inactivate RNases on glassware or metal, bake the glassware or metal for a minimum of 8
hours at 180°C.
Preventing Nucleic Acid Contamination
Since we will use our isolated RNA to synthesize cDNA for PCR amplification, it is important to
remove any residual nucleic acids from equipment that was used for previous nucleic acid
isolation.
PART Ia) PROTOCOL RNA ISOLATION:
1. KEEP EVERYTHING ON ICE & USE GLOVES
(minimize holding tubes in your hands since this warms
up the solution). Add 10 µl of chilled mercaptoethanol (-Me) to 1.5 ml of lysis buffer (in a v
vial). Vortex to mix. -Me is a reducing agent
(remember OILRIG?) that helps denature proteins as it
breaks disulfide bridges that maintains tertiary/quaternary
structure.
2. Weigh out 0.1 g of Xenopus ovary (this is about 100
oocytes) into a 1.5 ml v vial. Remove excess 0R-2
solution.
3. Add 600 µl of lysis buffer (with -Me) and homogenize
tissue/oocytes with a blue pestle.
4. Transfer into a 2 ml microfuge tube. Let the heavy
material settle to the bottom (wait 1 minute).
5. Avoiding the heavy material the bottom, transfer 600 µl of the homogenate to a
Prefilter Spin Cup (blue) that is seated in a 2-ml collection tube. Snap the cap from
the 2 ml centrifuge tube onto the blue spin cup by pushing on and stretching the
6
hinge. If you damage the lid (crimp edge of lid that is down into the blue spin
cup), it will leak- check edge of lid visually!!
6. Spin at “max speed” in a Fisher or BioRad mini-fuge
(2000 rcf, 6600rpm) or large microfuge (Eppendorf
runs at 16100 rcf or 13200 rpm) for 5 minutes.
7. Discard the blue spin cup and KEEP THE FILTRATE
(LIQUID) in bottom of receptacle (2ml centrifuge
tube).
8. Add 500 µl of 70% ethanol (in RNAse free water) to
the filtrate in 2 ml centrifuge tube. Cap the tube and
vortex for 10 seconds.
9. Transfer 600 µl of this mixture to an RNA Binding
Spin Cup (clear) that is seated in a fresh 2 ml
collection tube. Cap the spin cup as before-looking for any crimps.
10. Spin for 1 minute. RNA will stay in the clear RNA binding spin cup, contaminates
washed through cup into bottom of 2 ml tube.
11. Discard the filtrate (solution in bottom of tube). Transfer the remaining amount of
mixture from step 9 and spin for an additional minute (basically repeat steps 10 and
11).
12. Discard the filtrate – now all the RNA from the ovarian tissue is bound to the one
spin cup.
13. Add 600 µl of 1x Low-Salt Wash Buffer. Cap the spin cup and spin the sample for
1 minute.
14. Discard the filtrate. Replace the spin cup into the collection. Do not add anything to
the spin cup. The next step is a drying step to dry the column.
15. Spin the sample for 2 minutes. Discard filtrate.
16. In a separate microfuge tube, add 50 µl of DNase Digestion buffer to 5 µl of RNasefree DNase1 enzyme. Mix by pipetting gently up and down.
17. Add the 55 µl of DNase solution to the top of the fiber matrix (inside the spin cup).
7
18. Incubate the samples for 15
minutes in a 37 C water bath
(see photo) to digest the
DNA (leaving –
theoretically- only RNA).
19. Label clean 1.5 ml
microfuge tubes for final
RNA collection.
20. Add 600 µl of 1x High-Salt
Wash Buffer. Cap the spin
cup.
21. Spin the sample for 1
minute.
22. Discard the filtrate. Replace the spin cup into the collection.
23. Add 600 µl of 1x Low-Salt Wash Buffer. Cap the spin cup.
24. Spin the sample for 1 minute to remove contaminates.
25. Discard the filtrate. Replace the spin cup into the receptacle centrifuge tube.
26. Add 300 µl of 1x Low-Salt Wash Buffer. Cap the spin cup and spin the sample for
2 minutes.
27. Discard the filtrate.
28. Transfer the spin cup to the fresh 1.5 ml microfuge tube.
29. Add 30 µl of Elution Buffer to the center of the fiber matrix and incubate at room
temperature for 2 minutes. This elution buffer removes the RNA stuck to the fiber
matrix of the RNA binding spin cup.
30. Spin the sample for 1 minute. DON’T DISCARD FILTRATE IN BOTTOM OF
CENTRIFUGE TUBE—THIS IS RNA.
8
31. Wash spin cup but
repeating steps
29-30. You will
collect this second
elution in the
same microfuge
tube for a final
volume of ~60 µl
RNA.
PART Ib)
Quantitate the RNA
using the
Biophotometer.
Note Accurate
spectrophotometric
measurement requires
anOD260 ≥ 0.05.
We use the spec to
quantify RNA, DNA and protein. Nucleic acid (DNA, RNA) absorb light at 260 nm
(versus 276-280 for proteins). Double stranded DNA at a concentration of 50 µg/ml has
an OD260 of 1.0, 37µ/ml of single stranded DNA has an OD260 of 1.0, and 40 µg/ml of
single stranded RNA (the normal form—what we have just isolated) has an OD of 1.0.
Pure DNA preparations should have a 260/280 ration of 1.8, pure RNA preps should have
a ratio of 2.0. If you have a poor ratio, then you might have to reextract the DNA or
RNA prep with phenol:chloroform to remove protein impurities.
1. Press RNA (OD260 or A260) on the spectrophotometer. Blank the spectrophotometer
at 260 nm with an appropriate buffer (e.g., TE or 10 mM Tris, pH 7.5- near neutral pH) in
a UVette. (hit BLANK button- you should get a reading of 0.000 A).
2. Remove the blank UVette, press the DILUTION BUTTON, and type in µ of sample
(then hit ENTER), and the µl of diluent (hit ENTER). For example, 5 µl and 245 µl.
3. In a second UVette, dilute a sample of your RNA (1:50) by placing 5 µl into a cuvette
and then add 245 µl of TE buffer. Place a piece of laboratory film (e.g., Parafilm®
laboratory film- use the covered side of the Parafilm against your sample) over the top of
the cuvette and mix the sample well.
4. Take the reading. Record the A260, the A280, the A260/A280 ration and µg/ml
values.
5. Calculate the concentration of RNA. The conversion factor for RNA is 0.040 μg/μl
per A260 unit. Example: if the reading is 0.10- remember you programmed it to do the
dilution calculation for you, but check this…
Concentration of RNA= Absorbance260 × dilution factor × conversion factor
9
= 0.10×(DF: 250/5=50)× (1.0 OD260 for 0.040 μg/μl) = 0.2 μg/μl
for the concentration of RNA
5. Calculate the yield of RNA by mµltiplying the volume in microliters by the
concentration. For example, if you have 500 µl of RNA sample, you have 0.2 µg/µl x 500
µl = an RNA yield of 100 μg from about 100 mg of ovarian tissue (oocytes).
6. The ratio of the 260 nm measurement to the 280 nm measurement indicates purity.
Ratios of 1.8 to 2.1 are very pure. Lower ratios indicate possible protein contamination
(or low pH in the solution used as a diluent for the spectrophotometric readings).
Proteins absorb at 280 and nucleic acid (DNA, RNA) at 260 nm.
A second way of confirming that you have RNA is by running your sample on a gel. In
part II, we will be running our cDNA on a gel.
10
Lane
1
2
3
4
5
6
4.0 kb
2.3 kb
2.0 kb
28S
18S
Picture of total RNA isolation from Xenopus oocytes. Lane 1 and 6 contain DNA size
markers. A DNA standard that is “4.0 kb” means the nucleic acid is 4000 bases long.
Remmeber that the short/small nucleic acid moves faster and is closer to the bottom.
Lanes 2-5 contain 5 µl samples from four different RNA preparations. The Ethidium
bromide stains the nucleic acid, resulting in fluorescence under µultraviolet light. The
major RNA bands seen are the 28S and 18S rRNA transcripts (RNA that makes up the
ribosome; 28S and 18S are different sizes and the “S” stands for how fast it moves in
density gradient centrifugation- S=Svedberg unit). Note that the amount of mRNA is
relatively small and represented by faint minor bands. Some procedures only purify
mRNA (by using the polyA tail found on mRNA), not the total RNA that includes r and
tRNA.
PART Ic) PROTOCOL for RT-PCR from Total Oocyte RNA:
We will use Invitrogen’s One-Step SuperScript RT-PCR system. The most common
problem is contaminating DNA--DNA from previous procedures as DNA from your skin
(wear gloves) or aerosols of DNA solutions can get into pipettors, tubes, etc. Many labs
have a spot where the tubes for or from the PCR machine are opened- so that upon
opening, bits of DNA are released into the space around the PCR machine. Filters in
your pipette tips prevent aerosols enter in the pipette itself, and often separate pipettors
are used for PCR. Change your gloves frequently. Add DNA last to all reaction vials.
1. As before, SET UP All tubes and samples- ALL REACTIONS -ON ICE. Thaw
the template RNA, primers, 2X Reaction Mix, and RNase- free water. Place them on ice.
2. We want ~50 ng of RNA per reaction tube—you can calculate this amount from the
RNA quantification with the spec. We typically make a 1:100 dilution of your RNA
sample (mix 99 µl of RNase-free water with 1 µl of RNA).
3. You will set up the following PCR reactions:
TUBE 1.
phospholipase-C 
TUBE 2.
phospholipase-C  control no RNA
TUBE 3.
phospholipase-C  control no RT
1. Make up this MIXTURE #1 (keep on ice, in v vial):
11
component
volume (for 3 reactions)
RNase- free water
39 µl
2X Reaction Mix
75 µl
RNase inhibitor (RNasin;
from Promega)
5’ primer @ 5 uM
3µl
6 µl
3’ primer @ 5 uM
6 µl
2. To All 3 tubes, add 43 µl of the Mix #1.
Tube 1 gets
5 µl of 1:100 dilution of RNA
+ 43 ul of Mix #1 + 2 ul of
RT/Taq
Tube 2 gets
5 µl of RNA free water (no RNA) + 43 ul of Mix #1 + 2 ul of RT/Taq
Tube 3 gets
5 µl of 1:100 dilution of RNA
+ 43 ul of Mix #1 + 2 ul of Taq
alone (not RT/Taq)
MAKE SURE THAT YOU DO NOT CONTAMINATE STOCKS; USE A NEW
PIPETTE TIP EACH TIME!!
3. To tubes 1 and 3, add 5 µl of the 1:100 dilution
of RNA. Do not add RNA to tube 3, instead add
5 µl of RNase-free water. Tube 3 is your “no
template control” (RNA is the “template”) and is
used to evaluate the presence of nucleic acid
contamination in your RT-PCR reaction.
4. To tubes 1 and 2, add 2 µl of “RT-Taq” enzyme
mix (combination of reverse transcriptase and Tag
DNA polymerase). Do not add RT Taq enzyme
mix to tube 3, instead add 2 µl of Taq polymerase
WITHOUT RT. Tube 3 is your “no RT control”
to determine whether DNA is in your RNA
preparation.
5. Take your samples to the thermal cycler (also
called PCR machine). Transfer your entire ~50 µl
sample into the “PCR strip tubes” and place them into the small sample wells in the
front of rack. Mark the tab with your initial, and remember to place the samples
from left to right (on the left is sample from tube 1, then tube 2, 3).
6. Select the PCR program called RTPCR50 which automatically brings up the
following settings:
Reverse transcription (make cDNA from RNA):
45ºC for 30 min
12
Initial PCR activation step:
(heat denatures RT and other proteins)
3 step cycling—Denaturation temp(kills Taq):
Anneal temp (allows primers to bind)
Elongation temp (make DNA with Taq)
95º C for 2 min
94º C for 15 sec
50º C for 30 sec
68ºC for 1 min
40 cycles total
Final extension:
68ºC for 5 min
Cold hold (after ~4 hours, the PCR is finished, so this keeps the product cold for storage
overnight)
4ºC hold
Remove tubes from thermocycler and keep cDNA product at 2-8ºC.
Next class period you will run your samples (10 µl sample + 5 µl dye) on a 1% agarose
gel containing ethidium bromide, to visualize the RT-PCR resµlts.
Questions to think about.
1. If either of your negative controls show amplification products, what can you conclude
about the validity of your sample results?
2. What size fragment would you expect as a result of DNA amplification from genomic
DNA contamination? Think of this result relative to the size product you expect from the
RT-PCR amplification from RNA.
13
3. Discuss techniques that would actually quantify the amount of mRNA present. Some
cells may have a high level of gene activity, versus those cells with intermediate amount,
or zero amount of gene activity. See appendix below, use the web and your textbook.
Appendix: Quantify the amount of mRNA:
RT-PCR will confirm the presence of an mRNA, but is not considered a
quantitative method (like qRT-PCR) due to saturation of detection of the end product (in
other words, you may have different levels of gene activity and thus, different levels of
mRNA, but “regular” RT-PCR produces the same amount of product cDNA for different
starting amounts). However, RT-PCR can be designed to yield semi-quantitative
expression data, but this requires an elaborate replication (time) course analysis that we
will not perform.
For truly quantitative mRNA analysis qRT-PCR also called real time (or
quantitative) RT-PCR can be performed. qRT-PCR detects amplification of a gene
sequence as PCR amplification is occurring. This yields quantitative data, as levels of
amplification are being measured before the detection capacity reaches saturation. qRTPCR requires a highly specialized thermal cycler and fluorescent probes that allow for
real time amplified product detection.
Northern blot analysis, using labeled probes to detect mRNA levels can be
qualitative or quantitative. Northern blot analysis involves separating the RNA
molecµles by size in a denaturing agarose gel. The RNA is then transferred from the gel
onto a nylon membrane. A nucleic acid probe designed to be complementary to the RNA
sequence of interest is used to detect the presence and quantity of the RNA in the sample.
Northern blotting requires the use of either radioactive or chemical detection of the probe
and RNA hybridization.
Finally, with enough money, microarray or GeneChip analysis can be done to
look at expression of thousands of genes simultaneously in a semi-quantitative manner.
14