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) Measuringthe concentration of the environmental DNA
4) The qPCR assay of the standard and sample
5) Calculation of the qPCR amplification efficiency, and
6) The Answerthe concentration of the target gene using the standard curve.
Materials:
Qiagen plasmid prep kit / Clone with gene of interestPstI 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 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 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)
- 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.
- 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 ng/µl to 0.5 pg/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-Calculate amplification efficiency of the plasmid DNA (see below).
Picogreen assay of plasmid DNA standard:
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 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.
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).
/ 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).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 (
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.
/ 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
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/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, including the standard curve.
4- Label 4 microfuge tubes for the standard curve (A-D) and one tube for each of your samples.
5- Aliquot 150 µl of the kit DNA standards A-D to the tubes.
6- Aliquot 150 µl of TE buffer to microfuge tubes labeled for your samples.
7- Add 1.5 µl of sample DNA to the sample tubes containing TE buffer.
8- Add 150 µl of the stain solution to each standard and sample tube and mix.
9- Aliquot 100 µl 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 µl of sample was assayed compared to 150 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 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 µl qPCR reactions (1/4 reaction) as follows:
Prepare a fresh aliquot of Rox dye diluted 1 µl in 50 µl of TE buffer
µlComponent / 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
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
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 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/µl) by the concentration of DNA per liter to calculate the target gene abundance per liter of seawater.
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