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qPCR Protocol I: ABI instrument

Matt Cottrell and


Lisa Waidner
6/08
This protocol is for the ABI instrument. It differs slightly from that for the
RotorGene. Both protocols describe how to determine the abundance of a target gene in
environmental DNA using real time quantitative PCR (qPCR). The method consists of:
1) Preparation of a plasmid DNA standard
2) Removal RNA from template nucleic acid sample
3) Measuring the concentration of the environmental DNA
4) The qPCR assay of the standard and sample
5) Calculation of the qPCR amplification efficiency, and
6) The Answer the concentration of the target gene using the standard curve.

Materials:

Qiagen plasmid prep kit Clone with gene of interest


PstI restriction enzyme Picogreen DNA quantification kit
Black 96-well plate Fluorescence plate reader (Tim Targett's lab)
RNase One enzyme and buffer SYBR qPCR mix
qPCR thermal cycler (Cary lab) 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 g/ml in 50 l (total yield of about 6 g).
3- Digest an aliquot of the plasmid DNA with PstI
5 l of plasmid DNA (~0.5 g)
5 l of 10X buffer
40 l of water
1 l of PstI enzyme
4- Digest for 4 h 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)
i. Typical concentration after the PstI digestion is 15 ng/l if you
used the Qiagen plasmid prep kit and cut 5 l in a 50 l reaction.

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ii. Dilute the sample 30 fold (2 l of cut plasmid DNA + 58 l TE)
8- Prepare a 10-fold dilution series with 6 steps (a f) by diluting 20 l of plasmid
DNA with 180 l of TE. The resulting dilution series will span approximately 50
ng/l to 0.5 pg/l 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- Calculate amplification efficiency of the plasmid DNA (see below).

Picogreen assay of plasmid DNA standard:

1- Prepare a working stock (5 ng/l) of the picogreen standard kit DNA (100 ng/l)
Combine 10 l of the kit standard DNA + 190 l of TE buffer
2- Prepare the standard curve dilution series shown in Table 1 using 1.5 l tubes

Table 1. Dilution series of picogreen standard DNA for the standard curve.
Dilution pg/l x-fold from working stock TE (l) Working stock (l)
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 l 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 l of the kit DNA standards A-D to the tubes.
6- Aliquot 150 l of the plasmid dilutions (a-f) to microfuge tubes.
7- Add 150 l of the stain solution to each sample and blank and mix the tubes.
8- Aliquot 100 l of the stained sample to each of 3 wells in a black micro-titer
plate.

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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/l.

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).

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 will lower the efficiency and increase the slope of the
standard curve (Figure 2).

Figure 2. The slope increases with lower


efficiency because more cycles and thus
more amplification are required to reach
the fluorescence threshold (Ct).

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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 2.1 -3.10
100 2 -3.32
90 1.9 -3.59
80 1.8 -3.92
70 1.7 -4.34
50 1.5 -6.68

qPCR efficiency environmental DNA sample:

Amplification efficiency of environmental DNA can be assessed by determining the


Ct for different amounts of the DNA sample. It is common to use three DNA additions
differing by factors of ten prepared in a dilution series. Calculate the efficiency by
determining the slope of the regression of Ct versus the log of template DNA
concentration 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.

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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 m filtered seawater onto 25 mm dia, 0.2 m-pore-size
Durapore filters
2- Store the filters in CTAB buffer
3- Prepare the DNA following the chloroform procedure
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.
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:

1- Prepare a working stock (5 ng/l) of the standard kit DNA (100 ng/l)
Combine 10 l of the kit standard DNA + 190 l 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 l 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.

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5- Aliquot 150 l of the kit DNA standards A-D to the tubes.
6- Aliquot 150 l of TE buffer to microfuge tubes labeled for your samples.
7- Add 1.5 l of sample DNA to the sample tubes containing TE buffer.
8- Add 150 l of the stain solution to each standard and sample tube and mix.
9- Aliquot 100 l of the stained sample to each of 3 wells in a black micro-titer
plate.
10- Measure the fluorescence on the plate reader in Tim Targett's lab using the "Lisa
Fluor" program.
11- Be aware that the sample values must be multiplied by 100 because 1.5 l of
sample was assayed compared to 150 l of the standard.
12- If you are lucky you will now have 8.5 l of a template DNA at 5 ng/l that you
can dilute to 1 ng/l by adding 34 l of wate
qPCR of environmental DNA:
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 l qPCR reactions (1/4 reaction) as follows:
Prepare a fresh aliquot of Rox dye diluted 1 l in 50 l of TE buffer

l
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

Rox dye (1:50 in TE) 0.19 1.9 3.8 7.6 15.2


DNA 1

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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

Program the thermal cycler as suggested by Strategene:


PCR program for short targets (50 400 bp)
Cycles Duration of cycle Temperature (C)
1 10 minute 95
40 30 seconds 95
1 minute 55-60
1 minute 72

PCR program for long targets (400 900 bp)


Cycles Duration of cycle Temperature (C)
1 10 minute 95
40 30 seconds 95
1 minute 55-60
1.5 minute 72
1 3 minutes 72

A possible layout for 8 samples assayed in a 96-well plate is shown in Figure 4.

Figure 4. Layout of a 96-well plate for qPCR with plasmid standards, 8 environmental
samples and a negative control.
A B C D E F G H I J K L
1 30 pg plasmid 3 pg plasmid 300 fg plasmid 30 fg plasmid
2 3 fg plasmid 300 ag plasmid 10 ng Sample 1 1 ng Sample 1
3 0.1 ng Sample 1 Sample 2 ==> ==>
4 Sample 3 ==> ==> Sample 4
5 ==> ==> Sample 5 ==>
6 ==> Sample 6 ==> ==>
7 Sample 7 ==> ==> Sample 8

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8 ==> ==> Negative control

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.

Contamination and specificity:

Confirm that the negative control did not amplify and that the dissociation curve has a
single peak (Figure 5). If 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.
Using these unites the relative abundance of genes in a sample can be compared. For

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example, one could compare the abundance of a functional gene to 16S rRNA gene
abundance. Simply divide the gene copies/ng of one by the other to calculate the
functional gene:16S rRNA gene ratio.
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 (ng/l) by the concentration of
DNA per liter to calculate the target gene abundance per liter of seawater.