EGFP transgene quantification in genomic DNA
from mouse tissue
PROTOCOL FOR:
Real-time PCR to determine transgene copy number and to quantitate
the biolocalization of adoptively transferred cells from EGFP-transgenic
mice
Molishree U. Joshi1,2, H. Keith Pittman2, Carl E. Haisch2, and Kathryn M.
Verbanac1,2
1
Interdisciplinary Ph.D. Program in Biological Sciences and 2Department of Surgery,
East Carolina University, Greenville, NC, USA
BioTechniques 45:XXX-XXX (September 2008)
LEGEND
ATTENTION
* HINT
REST
This protocol describes a stepwise method to quantitate the biolocalized adoptively
transferred EGFP-transgenic cells by qPCR. C57BL/6-Tg(ACTbEGFP)10sb/J and
C57BL/6-Tg(UBC-GFP)30Scha/J mice were used as donors, and tumor-bearing wildtype C57BL/6J mice were used as recipients of adoptively transferred cells. All mice
were obtained from Jackson Laboratories.
REAGENTS
1 kb Plus DNA ladder (Gibco, Carlsbad, CA, USA)
200 proof ethanol (Sigma Aldrich, Saint Louis, MO, USA)
β-actin Gene Expression Assay: Mm00607939_s1 (Applied Biosystems, Foster City,
CA, USA)
Chloroform-Isoamyl alcohol 49:1 (Sigma Aldrich, Saint Louis, MO, USA)
DNA Exitus Plus (Applichem, Cheshire, CT, USA)
Mouse genomic DNA (Clontech, Mountain View, CA, USA)
Nuclease-free water (Ambion, Austin, TX, USA)
Phenol-Chloroform-Isoamyl alcohol 25:24:1 (Sigma Aldrich, Saint Louis, MO, USA)
Proteinase K (Gibco, Carlsbad, CA, USA)
RNaseA (Sigma, Saint Louis, MO, USA)
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SYBR-Green (Gibco, Carlsbad, CA, USA)
TaqMan® 2X Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA)
Tissue-Tek O.C.T compound (Sakura Finetek U.S.A., Torrance, California, USA)
Tris-EDTA (Gibco, Carlsbad, CA, USA)
PROCEDURE
ISOLATION OF GENOMIC DNA FROM MAMMALIAN TISSUE FOR qPCR
Precautions must be taken to avoid cross-contamination of samples. Pipets and
consumables should be regularly treated by UV irradiation to damage any
contaminating DNA and render it unable to be amplified. All surgical instruments
must be washed with mild soap, rinsed with distilled water and sprayed with 70%
ethanol after use. Work surfaces, equipment, and surgical instruments should be
routinely cleaned with bleach-containing disinfectants (e.g., Dispatch) and DNA
Exitus Plus, a commercial agent that degrades DNA into small fragments.
Instruments are subsequently autoclaved. Different sets of surgical instruments should
be dedicated to use with either green fluorescent protein (GFP) or wild-type mouse
strains and separate instruments should be used for each animal.
1. Surgically harvested tissue must be frozen immediately. Tissue should be a) wrapped
in aluminum foil and flash frozen in liquid nitrogen or b) embedded in specimen
molds with OCT and frozen using dry ice and acetone. All samples must be stored at
-70°C.
* A. FOR FLASH-FROZEN SAMPLES
A1. Prepare a foil spatula and a foil pouch with six layers. Immerse in liquid nitrogen
to chill.
A2. Weigh the frozen tissue (W1) in its original foil.
A3. Unwrap the foil and quickly but completely transfer the tissue, using the chilled
foil spatula, to the chilled foil pouch. Weigh original foil (W2) and calculate tissue
weight (Wt = W1–W2). Fold the pouch tightly and immerse in liquid nitrogen for
30 s.
A4. Remove pouch, place on bench, and immediately pulverize the tissue into powder
using a hammer. Work quickly and do not allow tissue to warm. This step is best
performed in a cold room.
A5. Suspend the powder in digestion buffer (3 ml / 50–100 mg of tissue). Vortex.
Samples should be pulverized extensively enough and quickly enough so that there
are no clumps after addition of digestion buffer. If there are clumps, longer digestion
times may be required. Continue with Step 2.
* B. FOR OCT SECTIONS
B1. Cut 10 μm–50 μm sections of optimum cutting temperature (OCT)-tissue block
and, using pre-chilled plastic tongs, collect the sections in cold polypropylene tubes
(Plastic tongs and 2 ml microfuge tubes must be placed in the -20oC cryostat unit to
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pre-chill.) The thickness and number of tissue sections can be varied to increase the
EGFP signal-to-background wild-type (noise) ratio. We recommend five 10 micron
sections for larger tissue portions (~10–15 mm diameter) and five 50 micron sections
for smaller tissue portions (≤8 mm diameter). Cap and store at -70°C until ready to
process.
B2. Fill the tube with room temperature (RT) PBS (pH 7, 320 osm) and incubate at
37°C for 15 min to remove OCT.
B3. Centrifuge at 500 × g for 10 min at RT and aspirate and discard the supernatant.
B4. Add digestion buffer (DB). The volume of DB will vary according to the number
of tissue sections. Minimum volume to be used is 500 μl. The volume of DB must be
increased if using more than the above recommended number or thickness of tissue
sections
Continue with Step 2.
2. Add 5 μl Proteinase K (reconstituted in water to 20 mg/ml) per 1 ml of tissue slurry in
DB.
3. Incubate the samples at 50°C for 3 h in a tightly capped tube. Tubes must be
vortexed 2–3 times during the incubation. The sample should be clear and viscous.
Tubes can be incubated up to 15 h if required.
4. Add 5 μl RNaseA (reconstituted in water to 5 mg/ml) per 1 ml DB.
5. Incubate at RT for 1 hour.
6. Add equal volume of Phenol-Chloroform-Isoamyl alcohol. Mix by inversion.
7. Centrifuge at 1500 × g, for 15 min, RT.
8. Transfer the top aqueous layer into a fresh tube. Add equal volume of ChloroformIsoamyl alcohol. Mix by inversion.
9. Centrifuge at 1500 × g, 15 min, RT.
10. Transfer the top aqueous layer into a fresh tube.
11. Add ammonium acetate (1/2 volume of supernatant). Mix by inversion.
12. Add 2 volumes of 100% ethanol. Mix by inversion.
Incubate at -20°C for 1 h (can be stored at -20°C for up to 2–3 days).
13. Centrifuge at 2000 × g for 20 min, 4°C.
14. Rinse the pellet with 70% ethanol. Centrifuge at 2000 × g for 5 min.
15. Decant ethanol and air-dry the pellet. (It is important to rinse well to remove residual
salt and phenol.)
16. Re-suspended DNA in 1× TE buffer (15–50 μl depending on the DNA pellet size)
until dissolved.
Optional: Leave it overnight at RT.
17. Heat at 50°C for 1 h to facilitate solubilization.
18. Check initial DNA concentration spectrophotometrically.
 Aliquot the DNA sample. Store samples at 4°C.
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* We recommend using OCT-frozen tissue sections if possible because sample recovery
is greater, discrete sections can be analyzed (for a higher signal:noise), and thin tissue
sections digest easily.
DNA yields averaged 2.5 μg /mg lung tissue and 3.8 μg / mg liver tissue. Although OCTembedded samples tended to have greater DNA yields, this difference was not
statistically significant in our hands. There was also no significant difference in the
quality of DNA obtained from flash-frozen versus OCT-embedded tissue, as evaluated by
OD260/280 and OD260/230 and integrity upon agarose gel electrophoresis. Finally, there was
no significant difference in the Ctβ-actin between flash-frozen or OCT-embedded tissue
samples when amount of input DNA was based on SYBR-green based fluorometric
assay.
DNA QUANTITATION BY SYBR GREEN FLUOROMETRIC ASSAY
 The SYBR-green based fluorometric assay must be used to accurately determine
DNA concentration of intact double-stranded DNA (see Troubleshooting section).
* Make sure that the genomic DNA is homogeneous by trituration and heating.
19. Make ten 2-fold dilutions for DNA standard curve using 1Kb Plus DNA ladder (8
ng/μl to 0.03 ng/μl) in 1× TE.
20. Dilute SYBR Green 1:1250 in 1X TE. Protect from light.
21. Dilute all samples in 1× TE based on the spectrophotometric concentrations such that
the dilutions fall within the range of the standard curve.
22. Heat all samples at 50°C for 10 min. Vortex and give a quick spin in a microfuge.
23. Add 100 μl of diluted samples to each well of a 96-well plate. Add 100 μl diluted
SYBR green solution to the wells (a multi-channel pipette is recommended). Each
plate must also include a blank (1× TE) and a positive control (mouse genomic
DNA).
24. Wrap the plate in aluminum foil. Incubate at RT for 10 min.
25. Read the plate on a fluorescent plate reader at 480/528 excitation/emission
wavelengths.
26. Using the linear equation from the standard curve, calculate the concentrations of
samples. Multiply the concentration by the dilution factor.
qPCR FOR QUANTITATION OF THE EGFP TRANSGENE
27. The concentrations of DNA from samples, negative control (DNA from wild-type
C57BL/6 mouse) and positive control (DNA from EGFP-transgenic mouse) must be
set to 10 ng/μl based on SYBR-green assay.
28. A standard curve is constructed in each assay using the pEGFP-N3 plasmid. The
dilutions are made by diluting plasmid in carrier DNA from wild-type C57BL/6 mice.
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The amount of carrier DNA in each dilution must be the same (10 ng/μl). The amount
of plasmid amount in standard curve ranges from 1 ng to 10 ag plasmid (see Table 1
in this protocol).
* The mass of one copy (or molecule) of the plasmid pEGFP-N3 was calculated from
the following formula:
m = mass of one copy = M / NA where
M = the molecular weight (Dalton or gram/mole) of the plasmid
(calculated using the base composition of the plasmid and MW of
nucleotides) = 3.09 × 106, and
NA = Avogadro′s number = 6.02 × 1023 copies (or molecules) /
mole.
Thus each molecule, or copy, of pEGFP-N3 has a mass of 5.1 attograms (ag) and 1 ng
of pEGFP-N3 has approximately 1.95 × 108 copies of EGFP transgene.
29. Prepare Master Mixes for amplifying EGFP transgene and housekeeping gene (βactin). See Recipes.
30. PCR for EGFP and housekeeping gene (β-actin) are run in separate wells. Add 15 μl
of each Master Mix to separate wells (see Example below). Add 10 μl DNA samples,
plasmid standard, positive control, negative control or 10 μl water (no template
control).
31. All reactions must be run for 50 cycles using standard ABI cycling conditions (2 min
at 50ºC, 10 min at 95ºC, and 50 cycles of 15 s denaturation at 95ºC and 1 min
annealing and extension at 60°C).
32. The negative sample and water should show no amplification.
33. Plot CT (cycle threshold) EGFP vs. log copy number for plasmid standard curve.
34. Use the linear equation from the plasmid standard curve to calculate log copy number
for DNA samples. This log copy number is then converted into copy number. The
number of EGFP transgene per 100 ng DNA is calculated in this way.
* The PCR efficiency (E) can be calculated from the slope of the standard curve. For a
linear equation y = mx + c, m is the slope. E = (10-1/m – 1) × 100.
In our experiment, the average linear equation was y = -3.31× + 40.01, and average
efficiency was 99.5% over at least 7 separate assays.
35. Depending upon the EGFP transgene copy number (number of EGFP transgene per
diploid cell) in the particular strain being used for adoptive transfer, EGFP cells per
100 ng DNA can be calculated (See Example below).
* The most concentrated plasmid dilutions should be aliquotted in 10 μl volume and
stored at -20°C.
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* Avoid more that 3–4 freeze-thaw cycles for primer and probes. Protect the probe from
light.
TABLES/FIGURES
Table 1: pEGFP Standard Curve for qPCR
P1
P2
P3
P4
P5
P6
Plasmid
Amount
10 ag
100 ag
1 fg
10 fg
100 fg
1 pg
Plasmid Copy
Number
2
20
200
2000
20,000
200,000
Plasmid Log
Copy Number
0.3
1.3
2.3
3.3
4.3
5.3
RECIPES
Recipe 1: Digestion buffer (DB)
Ingredient
NaCl
SDS
100× Tris-EDTA; pH 8
(1 M Tris-HCl, 0.1 M EDTA)
0.5 M EDTA (pH 8)
Milli-Q Water
TOTAL
Volume (ml)
Weight (gm)
Final Concentration
2.9
2.5
100 mM
17 mM
1×
5
(10 mM Tris-HCl, 1 mM EDTA)
25
470
500
25 mM
Recipe 2: 1 × TE
Ingredient
100× Tris-EDTA; pH 8
(1 M Tris-HCl, 0.1M EDTA)
Milli-Q Water
TOTAL
Volume (ml)
Final Concentration
1
1×
(10 mM Tris-HCl, 1 mM EDTA)
99
100
Recipe 3: Master Mix for qPCR (All PCR reagents purchased from Applied
Biosystems, unless otherwise noted.)
a) EGFP Master Mix
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Volume (l)
per reaction
well or tube
Final
Concentration
in Master Mix
Final
Concentration
in 25 l PCR
reaction (15 l
Master Mix + 10
l DNA)
12.5
0.25
0.25
0.75
1.25
15
1.67×
0.67 M
0.67 M
0.25 M
1×
0.4 M
0.4 M
0.15 M
Volume (l)
per reaction
well or tube
Final
Concentration
in Master Mix
Final
Concentration
in 25 l PCR
reaction (15 l
Master Mix + 10
l DNA)
12.5
1.25
1.67×
1.67×
1×
1×
TaqMan 2× Universal PCR Master Mix
40 M Primer 1 (5′-ccacatgaagcagcaggactt-3′)
40 M Primer 2 (5′-ggtgcgctcctggacgta-3′)
5 M Probe (6FAM-ttcaagtccgccatgcccgaa-TAMRA)
Nuclease free water
TOTAL
b)
β-actin
Master Mix
TaqMan 2× Universal PCR Master Mix
20 × Gene Expression Assay (Mm00607939_s1)
(Includes primers and probe)
Nuclease free water
TOTAL
1.25
15
* Prepare fresh Master Mix for each assay. Prepare Master Mix for 1-3 extra tubes/ wells
than the required number of tubes / wells. All reagents and Master Mix must be on ice. The
probe must not be exposed to light.
EXAMPLE
EGFP Master mix (15ul)
+ DNA (10l)
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-actin Master mix (15l)
+ DNA (10l)
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

Plasmid standard curve linear equation: y =-3.31× + 40.01, where y is CT EGFP
and x is Log copy number.
Cell for adoptive transfer harvested from C57BL/6-Tg(ACTbEGFP)10sb/J or
C57BL/6-Tg(UBC-GFP)30Scha/J; both strains have two copies of EGFP
transgene per diploid cell.
Sample
No template control:
water
Negative control:
Wild-type DNA
Positive control:
EGFP transgene DNA
Sample 1*
Sample 2*
Sample 3*
Sample 4*
Average CT
β-actin
Average CT
EGFP
Log Copy
Number
per 100
ng
Copy
Number
per 100
ng
Transgenic
cells per
100 ng
Undetected
Undetected
0
0
0
23.5
Undetected
0
0
0
23.5
25
4.5
34,256
17,128
23.4
35
1.5
33
16
23.5
33
2.1
132
66
23.7
39
0.3
2
1
23.2
Undetected
0
0
0
*Metastatic tumors from wild-type mice with metastases after adoptive transfer of EGFPVEC.
TROUBLESHOOTING
Negative or no template control samples result in positive amplification for EGFP
transgene.
Cause
Sample crosscontamination while
pipeting liquids
BioTechniques Protocol
Solution
Take precautions while handling plasmid, negative, positive
and experimental samples.
1. Change gloves frequently.
2. Pipet negative control samples first, then experimental
samples, then positive control samples.
3. Keep tube lids closed and space tubes apart,
4. Work in dead air environment and dedicated workstation.
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Contaminated surgical
instruments
Contaminated reagents
Contaminated equipment
or work area
Wash with soap and distilled water. Generously spray DNA
Exitus Plus and allow drying. Bag the instruments and
autoclave the surgical instruments.
Re-assay freshly prepared DNA dilutions for EGFP
amplification in triplicate; if positive, discard DNA sample
but if negative then it could be a technical error while
pipeting.
Clean all areas thoroughly, as described. We recommend
routine wipe tests to monitor surfaces (benches, centrifuges,
door and equipment handles, etc.) for contamination. This
should be done monthly or quarterly, depending on the
frequency of assays, as described in the article.
Discrepant CT values for housekeeping gene between different tissues
Cause: UV spectrophotometry can overestimate DNA concentration because
contaminating proteins and organic compounds may also absorb ultraviolet light (1). We
have found that DNA from certain tissue samples (e.g., liver) have a high degree of
contamination by proteins or organic compounds. This problem was undetected by
OD260:OD280 ratios, which were comparable in all tissues, but was evident upon agarose
gel electrophoresis (less DNA mass observed in liver samples) and evident by PCR for
the housekeeping control gene (less amplimer / higher CT observed in liver samples). We
observed a 4-fold difference in DNA concentration between the two methods for liver
tissue.
Solution: SYBR-green based fluorometric assay was the optimal method for quantitation
of intact double-stranded DNA greater than 200 bp; the dye intercalates between AT-rich
helices (2). We initially estimate the DNA concentration spectrophotometrically (e.g.,
using a NanoDrop) to estimate the dilution factor for the SYBR-green assay.
EQUIPMENT
ABI Prism (Applied Biosystems, Foster City, CA, USA)
BioTek plate reader (BioTek, Winooski, VT, USA)
REFERENCE
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1. Sambrook, J. and D.W. Russell. 2001. Commonly used techniques in molecular
cloning, Appendix 8, p. A8.1-A8.55. In Molecular Cloning, 3rd ed. CSH Laboratory
Press, Cold Spring Harbor, NY.
2. Leggate, J., R. Allain, L. Isaac, and B.W. Blais. 2006. Microplate fluorescence assay
for the quantification of double stranded DNA using SYBR Green I dye. Biotechnol.
Lett. 28:1587-1594.
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