Introduction to RNA Quantification
Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial
distribution of RNA expression. A number of widely used procedures exist for detecting and
determining the abundance of a particular mRNA in a total RNA sample. The most popular methods
are: Northern blot analysis, Nuclease Protection Assays (NPA), in situ hybridization, and Reverse
Transcription-Polymerase Chain Reaction (RT-PCR).
In theory, each of these techniques can be used to detect specific RNAs and to determine their
expression level. However, each method has inherent advantages and/or limitations. In general,
Northern analysis is the only method that provides information about transcript size, whereas NPAs are
the easiest way to simultaneously examine multiple messages. In situ hybridization is used to localize
expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method
for detecting and quantitating gene expression. Northern blotting and nuclease protection assays are
also limited by their sensitivity. Ribonuclease protection assay is the most sensitive non-PCR based
mRNA detection and quantitation procedure but RT-PCR is significantly more sensitive than this; it is
capable of detecting moderately expressed transcripts from a single cell.
PCR
Polymerase chain reaction has rapidly become one of the most widely used techniques in molecular
biology and for good reason: it is a rapid, inexpensive and simple means of producing relatively large
numbers of copies of DNA molecules from minute quantities of source DNA material--even when the
source DNA is of relatively poor quality.
PCR mimics the process by which most organisms copy their DNA, only it does it in a test tube. When
any cell divides, enzymes called polymerases make a copy of all the DNA in each chromosome. The
first step in this process is to "unzip" the two DNA chains of the double helix. As the two strands
separate, DNA polymerase makes a copy using each strand as a template.
To copy DNA, polymerase requires two other components: a supply of the four nucleotide bases
(ATGC) and something called a primer. DNA polymerases, whether from humans, bacteria, or viruses,
cannot copy a chain of DNA without a short sequence of nucleotides to "prime" the process, or help the
polymerase attach to the DNA. In vivo, the cell has another enzyme called a primase that actually
makes the first few nucleotides of the copy. This stretch of DNA is called a primer. Once the primer is
made, the polymerase can take over making the rest of the new chain.
A PCR vial contains all the necessary components for DNA duplication: a sample of total DNA, large
quantities of the four nucleotides, large quantities of primers that recognize specific region of the gene
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of interest, and DNA polymerase. The polymerase used is the Taq polymerase, named for Thermus
aquaticus, from which it was isolated.
The three parts of the polymerase chain reaction are carried out in the same vial, but at different
temperatures. The first part of the process separates the two DNA chains in the double helix. This is
done simply by heating the vial to 90-95 degrees centigrade (about 165 degrees Fahrenheit) for 30
seconds.
But the primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 55
degrees C (about 100 degrees F). At this temperature, the primers bind or "anneal" to the ends of the
DNA strands. This takes about 20 seconds.
The final step of the reaction is to make a complete copy of the templates. Since the Taq polymerase
works best at around 72 degrees C (the temperature of the hot springs where the bacterium was
discovered), the temperature of the vial is raised. The Taq polymerase begins adding nucleotides to the
primer and eventually makes a complementary copy of the template. This completes one PCR cycle.
Figure 1: PCR steps.
The three steps in the polymerase chain reaction - the separation of the strands, annealing the primer to
the template, and the synthesis of new strands - take less than two minutes. Each is carried out in the
same vial. At the end of a cycle, each piece of DNA in the vial has been duplicated.
But the cycle can be repeated 30 or more times. Each newly synthesized DNA piece can act as a new
template, so after 30 cycles, 1 million copies of a single piece of DNA can be produced (figure 2).
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Figure 2
Theoretically, the amplification for a given number of cycles can be approximated using the formula:
Y = A.2n
where A: initial number of copies; n = number of cycles.
However, there are many details that this idealization ignores. The first simplification is to assume that
the actions of denaturing, annealing, and extension occur only when the reaction is at the proper
temperature for them to occur and that they occur to completion at that temperature. In fact, these are
all kinetic reactions occurring simultaneously with very temperature dependent rates. This of course
means that replication need not be two-fold per cycle; it can be more or less.
The next assumption is that the reaction contains a constant (and large) primer, enzyme, and dNTP
concentration in every cycles. The fact is that all this components are quicklly used and trerefore their
concentracion decrease as cylces increases. If one begins a reaction with 1000 template molecules and
1uM primer molecules, in only 18 rounds of amplification all the primer will be depleted from the
reaction. The enzyme, usually chosen because of its ability to withstand denaturing temperatures still
suffers from denaturation with a half-life ranging from 130 minutes at 92.5 C to only 6 minutes at 97.5
C. Therefore, as the number of template molecules approaches the number of enzymes still active, not
all template-primer duplexes will be extended. Of course, limiting the amount of dNTPs will also
decrease the effective activity of the enzyme and their complete depletion will halt the reaction. For
these reasons, the amounts of primer, enzyme, and dNTPs must be chosen based on the amont of
desired terminal DNA product concentration.
Considering these effects, we can asume that the early cycles of PCR are characterized by an
exponential increase in target amplification, because during this period there is still excess of reactants
and the enzyme is fully active. When this conditions are no longer present the accumulation of product
amplification generally drops off dramatically and the product stops accumulating exponentially until a
plateau is reached and there is little or no net increase in PCR product. Conditions that contribute to
reach this plateau are: - The utilization of substrates, either primer or dNTPs, - Stability of the reactants
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(decreased activity of the enzyme), - End product inhibition (PCR product competes with primer during
annealing), and - Incomplete denaturation at higher product concentration.
Ideal PCR
1.E+09
Signal (Log)
Actual PCR
1.E+06
1.E+03
10
20
Cycle Number
30
Figure 3: PCR effect on number of cycles
Relative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Quantitation of transcription via RT-PCR can be approached using either relative RT-PCR or a
competitive RT-PCR strategy. Theoretically, relative quantitation by RT-PCR can grossly estimate
differences in abundance of a particular transcript between samples if the following conditions are met:
•
The amount of RNA used in the initial cDNA synthesis reaction is precisely controlled.
•
The amount of cDNA used in the PCR is precisely controlled.
•
The number of PCR cycles necessary to generate enough detectable products is not beyond the
exponential phase of the PCR.
Therefore, for relative RT-PCR data to be meaningful, the PCR reaction must be terminated when the
products from both the internal control and the gene of interest are detectable and are being amplified
within linear range. The linear range of amplification occurs in the PCR cycles when reaction
components are still in excess and the PCR products are accumulating at a constant rate. On the other
hand, the amount of PCR product measured beyond the exponential phase (plateau) is very sensitive to
slight variations in initial amplification target which makes quantitation in this zone unreliable.
To determine linear range, a PCR reaction is performed at increasing cycle numbers and resolved by
electrophoresis. The products are visualized by ethidium bromide staining and quantitated with a
computer software. Cycle number can be plotted against the signal obtained in order to identify the
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exponential (linear) range and the plateau phase. If cycle numbers is plotted against the log of the
signal, a straight line should be obtained for samples in linear range amplification.
One should be also aware that a minimum number of cycles may be necessary to produce a visible
band, which depends largely on the starting concentration of the target DNA and in the detection
method used.
Bench work
Reagents
1. DNA template: Product of reverse transcriptase (RT) reaction (cDNA). We generally performed RT with 1
ug of total RNA which is diluted after RT to 100 ul given approx. 50 nanograms each 5 ul.
2. Primers: Primers are re-suspended in ddH20 at 100 picomols/ul.
3. Buffer: Buffer is generally provided with the enzyme as a 10x stock.
4. Taq DNA polymerase: Taq is used at 2.5 U per reaction. Takara is provided at U/ul.
5. Nucleotides: 2.5 mM each dNTP. dNTPs is provided with the enzyme as a 10 x stock
6. Water: Water should be autoclaved and used solely for PCR. Milli-Q water is fine for PCR. It can be
aliquotted into 1ml volumes and kept separate from DNA and other sources of contamination. Each aliquot
should be discarded following single use.
7. Mineral Oil: In some instruments mineral oil must be added to prevent evaporation of the sample.
Experiment 1: Determination of exponential phase of amplification for the ribosomal protein L19
that is used as internal control.
Setting the Reaction
WORK ALL THE TIME ON ICE
1. Label PCR tube according the following protocol:
Sample A tubes: A1, A2, A3, A4, A5, A6, A7 and sample B tubes: B1, B2, B3, B4, B5, B6, B7.
2. Make Master MIX of reagents using DNA-free pipette.
Reagents
ul per RXN
PCR Buffer 10x
dNTPs
L19 Anti-sense Primer
L19 Sense Primer
Enzyme Taq Pol
ddH2O
4
2
0.4
0.4
0.2
28
x RNXs
3. Vortex and centrifuge the MIX
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4. Dispense 35 ul of Master MIX into individual tubes.
5. Add 5 ul of template DNA to each tube. (It is essential in this step to prevent any carry over contamination).
6. Add H2O to tube 7 as negative control.
7. Overlay reactions with 50 ul of mineral oil to ensure no evaporation occurs: this changes reactant
concentrations.
8. Vortex and briefly centrifuge.
9. Place samples in PCR machine.
10. Star a program with Pre-denaturation phase of 94 C for 1 min
11. Perform cycles with the following characteristics.
Denaturation
94C
40 sec
Annealing
63C
40 sec
Extension
72C
40 sec
12. Take the tube out of the PCR machine at the end of the extension phase at following cycles (place the tube
on ice).
Tube
1
2
3
4
5
6
7
Cycle #
10
15
20
25
30
35
35
Experiment 2: Relative quantitation of the Nur77 mRNA levels.
1. Label PCR tube according the following protocol:
Control
PGF2α 0.1 mM
PGF2α 1 mM
PGF2α 5 mM
H2O
2. Make Master MIX of reagents using DNA-free pipette.
Reactive
ul per RXN
PCR Buffer 10x
dNTPs
L19 Antisense Primer
L19 Sense Primer
Nur77 Anti-sense Primer
Nur77 Sense Primer
Enzyme Taq Pol
Complete with ddH2O to 35 ul
4
2
0.4
0.4
0.4
0.4
0.2
x RNXs
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3. Vortex and centrifuge the MIX
4. Dispense 35 ul of Master MIX into individual tubes.
5. Add 5 ul of template DNA to each tube. (It is essential in this step to prevent any carry over contamination).
6. Add H2O to tube A7 as negative control.
7. Overlay reactions with 50 ul of mineral oil to ensure no evaporation occurs: this changes reactant
concentrations.
8. Vortex and briefly centrifuge.
9. Place samples in PCR machine.
10. Star a program with Pre-denaturation phase of 94 C for 1 min
11. Perform 21 cycles with the following characteristics.
Denaturation
94C
40 sec
Annealing
63C
40 sec
Extension
72C
40 sec
12. Take the tube out of the PCR machine at the end of the extension phase at following cycles (place the tube
on ice).
Detecting Products.
AT THIS STEP WORK IN AN AREA AWAY FROM WHERE YOU USUALLY SETUP THE PCR
REACTION!
1. Add 5 ul of 10 x gel loading buffer to each sample.
2. Load 5 – 15 ul of sample into wells of 2.0% submarine polyacrylamide gel made up in TBE, preferably
containing 50 ng/ml ethidium bromide (ethidium bromide is toxic please wear glove all the time).
3. Load also a DNA markers going from 2kb down to 100 bp.
4. Run at 80 -120 volts (not too slow or small products diffuse; not too fast or bands smear) until bromophenol
blue reaches 2/3 of gel.
5. View on UV light box.
6. Take Picture with Kodax 1D software and quantify each band (Net intensity).
7. Plot cycle number vs Net intensity and cycle number vs Log of net intensity for the Exp 1. For exp 2
calculate the relative expression of Nur77 by dividing Nur77 intensity/L19 intensity.
Conclusions.
You have to present a graph with its respective conclusion and discuss the results with the others
groups.
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