qPCR Protocol II
Cottrell
8/5/09
This protocol is for the RotorGene qPCR machine. It differs slightly from the
protocol for the ABI machine. Both have the same goal of describes determining the
abundance of a target gene in environmental DNA using real time quantitative PCR
(qPCR).
Follow the steps below to prepare a plasmid DNA standard, measure the
concentration of the environmental DNA, perform the qPCR assay of the standard and
sample, calculate the qPCR amplification efficiency and finally calculate abundance of
the target gene using the standard curve.
Materials:
Qiagen plasmid prep kit
Restrictionase to linearized plasmid (e.g. PstI )
Black 96-well plate
RNase One enzyme and buffer
qPCR thermal cycler
Clone with gene of interest
Picogreen DNA quantification kit
Fluorescence plate reader
SYBR qPCR mix
Environmental DNA sample
Procedures:
Plasmid DNA standard of the target gene:
1- Prepare the plasmid using the Qiagen plasmid prep kit.
2- Measure concentration of the plasmid DNA using A260 absorbance. Typical
concentration is 150 µg/ml in 50 µl (total yield of about 6 µg).
3- Digest an aliquot of the plasmid DNA with PstI
5 µl of plasmid DNA (~0.5 µg)
5 µl of 10X buffer
40 µl of water
1 µl of PstI enzyme
4- Digest for 4 h or over night at 37 °C
5- Stop the enzyme by incubating at 65° C for 20 min.
6- Run an agarose gel of cut and uncut plasmid DNA to confirm the digest.
7- Dilute the linearized plasmid DNA standard (qPCR standard for your gene)
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i. Typical concentration after the PstI digestion is 15 ng/µl if you
used the Qiagen plasmid prep kit and cut 5 µl in a 50 µl reaction.
ii. Dilute the sample 30 fold (2 µl of cut plasmid DNA + 58 µl TE)
8- Prepare a 10-fold dilution series with 6 steps (a – f) by diluting 20 µl of plasmid
DNA with 180 µl of TE. The resulting dilution series will span approximately 50
pg/µl to 0.5 fg/µl of plasmid DNA.
9- Check the DNA concentration in the dilution series using the picogreen assay (see
below)
10- Test the PCR primers with this plasmid DNA dilution series.
11- Measure the amplification efficiency of the plasmid DNA (see below).
Picogreen assay of the plasmid DNA:
1- Prepare a working stock (5 ng/µl) of the picogreen standard kit DNA (100 ng/µl)
 Combine 10 µl of the kit standard DNA + 190 µl of TE buffer
2- Prepare the standard curve dilution series shown in Table 1 using 1.5 µl tubes
Table 1. Dilution series of picogreen kit standard DNA for the standard curve.
Dilution
pg/µl
x-fold from working stock
TE (µl)
Working stock (µl)
A
500
10x
450
50
B
100
50x
490
10
C
50
100x
495
5
D
5
100x from A
495
5 from A
Blank
0
none
500
none
3- Prepare a 200-fold dilution of the picogreen stain. 10 µl stain + 2 mL of TE
buffer is enough to measure the DNA concentration in 7 samples, including the
standard curve.
4- Label 4 microfuge tubes for the standard curve (A-D) and one tube for each of
your samples (a-f).
5- Aliquot 150 µl of the kit DNA standards A-D to the tubes.
6- Aliquot 150 µl of the plasmid dilutions (a-f) to microfuge tubes.
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7- Add 150 µl of the stain solution to each sample and blank and mix the tubes.
8- Aliquot 100 µl of the stained sample to each of 3 wells in a black micro-titer
plate.
9- Measure the fluorescence on the plate reader in Tim Targett's lab using the "Lisa
Fluor" program.
10- The "Lisa fluor" program output is the plasmid DNA concentration in pg/µl.
qPCR efficiency – plasmid DNA standard:
The amount of DNA in the PCR reaction doubles with every amplification cycle of a
100% efficient reaction. Therefore doubling the amount of template DNA added to the
reaction will reduce the Ct value by 1 cycle. Plotting Ct versus log10 of gene copies in
the template DNA yields a line with a slope of -3.32 for a reaction with 100% efficiency
(Figure 1). The y-intercept is the number of cycles needed to amplify a single target
molecule.
Figure 1. The efficiency of a qPCR
reaction is 100% when the DNA doubles
with each cycle. Doubling the gene copies
added as template DNA reduces the Ct
value by 1 cycle.
Slope = -1/log10 2 = -1/0.301 = -3.32
Inhibitors in the template DNA, poor primer annealing, etc. will lower the efficiency
and increase the slope of the standard curve (Figure 2).
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Figure 2. The slope increases with lower
efficiency because more cycles are required
to achieve the amount of amplification
needed to reach the fluorescence threshold
(Ct).
The slope of the linear regression of Ct vs log10 of gene copies can be used to
calculate the PCR efficiency from equation 1.
(1) Efficiency % = 100 x (10(-1/slope) – 1 )
A range of efficiencies is shown in Table 2.
Table 2. A good qPCR run will have an efficiency ranging from 90%-110%. The
relationship between slope and efficiency follows the equation:
Slope = -1 x 1/log10 Fold increase per cycle
(http://tools.invitrogen.com/content/sfs/appendix/PCR_RTPCR/Important%20Parameters%20of%20qPCR.
pdf)
Efficiency %
Fold increase per cycle
Slope
110
100
90
80
70
50
2.1
2
1.9
1.8
1.7
1.5
-3.10
-3.32
-3.59
-3.92
-4.34
-6.68
qPCR efficiency – environmental DNA sample (optional):
Amplification efficiency of environmental DNA can be assessed by determining the
Ct for different amounts of the DNA sample. In an ideal world the efficiency would be
determined for every environmental sample, but it is often not practical when assaying
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many samples or when abundance of the target gene is low. Test three DNA additions
differing by factors of ten prepared in a dilution series and calculate the efficiency by
determining the slope of the regression of Ct versus the log of template DNA amount
added. For a 10-fold dilution series of DNA with three steps the x-axis would be 0, -1
and -2, corresponding to the 1, 0.1 and 0.001-fold dilutions. For example, if the
undiluted DNA sample has a Ct of 20, then the 10 and 100-fold dilutions would have Ct
values of 23.32 and 26.64, respectively (Figure 3).
It probably takes some trial and error to find a dilution series that gives results that are
on scale (Ct between 10 and 30). Table 3 summarizes some possible outcomes for
various template DNA additions.
Figure 3. Example data for an
environmental DNA sample that amplifies
with 100% efficiency. Three 10-fold
dilutions of the template DNA were
assayed. The highest concentration of
DNA had a Ct of 20, so the 10-fold and
100-fold dilutions had Ct values of 23.32
and 26.64, respectively.
Template environmental DNA preparation:
1- Filter 1 to 2 liters of 0.8 µm filtered seawater onto 25 mm dia, 0.2 µm-pore-size
Durapore filters
2- Store the filters in CTAB buffer
3- Prepare the DNA following the chloroform procedure, using two chloroform
extractions and being sure not to carry over any chloroform into the next steps.
4- Measure the concentration of DNA using the picogreen assay as described below.
Table 3. Ct values for 10-fold dilution series of DNA (100% efficiency). Values are
given for undiluted DNA yielding Ct values of 10, 20 and 30.
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DNA dilution
Ct
1
10
20
30
0.1
13.32
23.32
33.32
0.01
16.64
26.64
36.64
Picogreen assay of template environmental DNA (volumes adapted to Rotor-Gene):
1- Prepare a working stock (5 ng/µl) of the standard kit DNA (100 ng/µl)
 Combine 10 µl of the kit standard DNA + 190 µl of TE buffer
2- Prepare the standard curve dilution series shown in Table 1.
3- Prepare a 200-fold dilution of the picogreen stain. 10 µl stain + 2 mL of TE
buffer is enough to measure the DNA concentration in 7 samples and the standard
curve.
4- Label 4 microfuge tubes for the standard curve (A-D) and one tube for each of
your samples.
5- Aliquot 80 µl of the kit DNA standards A-D to the tubes.
6- Aliquot 76 µl of TE buffer to microfuge tubes labeled for your samples.
7- Add 4 µl of sample DNA to the sample tubes containing TE buffer.
8- Add 80 µl of the stain solution to each standard and sample tube and mix.
9- Aliquot 50 µl of the stained sample to each of 3, 0.2 ml Rotor-Gene tubes.
10- Measure the fluorescence in the Rotor-Gene instrument.
11- Be aware that the sample values must be multiplied by 20 because 4 µl of sample
was assayed compared to 80 µl of the standard.
12- If you are lucky you will now have 8.5 µl of a template DNA at 5 ng/µl that you
can dilute to 1 ng/µl by adding 34 µl of water.
qPCR of environmental DNA:
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A qPCR experiment can include all of the necessary standard curves, controls and
samples needed for a clear interpretation of the following data:
1- Copies of the target gene per ng of environmental DNA
2- Plasmid control amplification efficiency
3- Environmental DNA amplification efficiency
4- Contamination of the PCR reaction
Set up 12.5 µl qPCR reactions (1/4 reaction) as follows:
Prepare a fresh aliquot of Rox dye diluted 1 µl in 50 µl of TE buffer
µl
Component
1X
10X
20X
40X
80X
Water
4.5
45
90
180
360
SYBR mix
6.25
62.5
125
250
500
Primer F*
0.25
2.5
5
10
20
Primer R*
0.25
2.5
5
10
20
DNA
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*Lower primer concentration may be necessary to minimize the formation of primer
dimmers during the amplification.
Program the thermal cycler as suggested in the following example:
PCR program for short targets (50 – 400 bp)
Cycles
Duration of cycle
Temperature (°C)
1
10 minute
95
40
15 seconds
95
45 seconds
55-60
15 seconds
72
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Data analysis:
Calculate the linear regression of Ct versus log gene copy number in the plasmid
DNA standards. Use this relationship to calculate the gene copy number in the
environmental DNA from the Ct value for the environmental sample.
Calculate the efficiency of amplification of the plasmid DNA and environmental
DNA as discussed above.
The molecular weight of one bp = 660, so 50 pg of a plasmid corresponds to 9.2 x 106
copies of a typical 1 kb target gene cloned in the 3.9 kb TOPO cloning vector (Table 4).
Table 4. Mass and copy number for a typical plasmid control dilution series.
Plasmid DNA (mass)
Gene copies
50 pg
9,200,000
5 pg
920,000
500 fg
92,000
50 fg
9,200
5 fg
920
500 ag
92
Contamination and specificity:
Confirm that the negative control did not amplify and that the dissociation curve has a
single peak (Figure 5). A broad second peak at 65°C often indicates primer dimmer. If
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the dissociation curve has more than a single peak run the amplification product on an
agarose gel to determine that the multiple peaks truly indicate non-specific amplification.
Multiple peaks do not necessarily indicate non-specific amplification because PCR
products that possess AT-rich regions can melt non-uniformly, generating multiple peaks
in the dissociation curve.
Figure 5. Dissociation curve of PCR
products appear as a single peak, indicating
the amplified genes have similar sequences
(G+C content). PCR products with very
different sequences will melt at different
temperatures and yield multiple peaks.
Gene abundance per ng of DNA, per 16S rRNA gene and per liter of seawater:
Results of qPCR analysis have units of target gene copies/ng of environmental DNA.
It is most straightforward to compare the abundance of a gene in different DNA samples
by comparing the gene copies/ng of DNA. The only assumptions are that amplification
efficiency is the same in the two samples and that the two samples contain the same
amounts of non-target DNA. We typically target microbes, so non-target DNA would
include detrital DNA or eukaryotic DNA, for example. The same approach can be used
to compare the abundance of a functional gene to the 16S rRNA gene abundance. Simply
divide the gene copies/ng of the functional gene by the copies/ng of the 16S rRNA gene
to calculate the functional gene:16S rRNA gene ratio.
It is also possible to determine the abundance of one gene relative to another, such as
a functional gene abundance relative to 16S rRNA gene abundance, by running the qPCR
for the two genes using the same aliquot of the diluted environmental DNA. The amount
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of DNA in the qPCR assay does not come into play using this approach. Use equation 2
to calculate the ratio of functional gene abundance/16S rRNA gene abundance by
comparing the Ct values obtained using the same amount of environmental DNA assayed
in the two qPCR assays.
(2) Functional gene /16S rRNA gene = 1/ ((Ct functional gene – Ct 16S rRNA gene) x 2)
If there are no other gene abundance data available for the sample it is possible to
estimate the percentage of the bacteria in the sample that carry the target gene by
multiplying the gene copies/ng DNA by an estimate of the average genome size in the
sample. A good estimate of average marine bacterial genome size is 3.5 fg. Then divide
by the expected gene copy number in the targeted genomes. For a single copy gene
divide by 1 or for a multi-copy gene such as the 16S rRNA gene you might divide by 1.5
or 2.
Determining the absolute abundance of gene copies per volume of seawater requires
an estimate of the amount of extracted DNA per volume of seawater. Divide the amount
of DNA extracted by the volume of seawater filtered to estimate the concentration of
DNA per liter. Then divide the target gene abundance (copies/ng) by the concentration
of DNA per liter to calculate the target gene abundance per liter of seawater. Of course
this approach requires assumptions about DNA extraction efficiency.
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